Valproic acid for the treatment or prevention of pathological conditions associated with excess fibrin deposition and/or thrombus formation

ABSTRACT

There is herein provided valproic acid, or a pharmaceutically acceptable salt, thereof, for use in treating or preventing a pathological condition associated with excess fibrin deposition and/or thrombus formation, wherein said treatment comprises treating a patient with valproic acid, or a pharmaceutically acceptable salt thereof, in a specific manner, and formulations for use or designed for use in such treatments.

STATEMENT OF PRIORITY

This application is a 35 U.S.C. § 371 national phase application ofInternational Application Serial No. PCT/GB2015/052950, fled Oct. 8,2015, which claims the benefit, under 35 U.S.C. § 119 (a) of UnitedKingdom Patent Application No. 1417828.9, filed Oct. 8, 2014, the entirecontents of each of which are incorporated by reference herein.

FIELD OF THE INVENTION

The present invention generally relates to new medical uses, methods oftreatment and pharmaceutical compositions. More specifically, it relatesto the use of valproic acid (VPA), and pharmaceutically acceptable saltsthereof, in the treatment or prevention of thrombus formation and inimproving or normalizing endogenous vascular fibrinolysis.

BACKGROUND OF THE INVENTION

The listing or discussion of an apparently prior-published document inthis specification should not necessarily be taken as an acknowledgementthat the document is part of the state of the art or is common generalknowledge.

Cardiovascular disease is the leading cause of morbidity and mortalityin the western world and during the last decades it has also become arapidly increasing problem in developing countries. An estimated 80million American adults (one in three) have one or more expressions ofcardiovascular disease (CVD), such as hypertension, coronary heartdisease, heart failure, or stroke. Mortality data show that CVD was theunderlying cause of death in 35% of all deaths in 2005 in the UnitedStates, with the majority related to myocardial infarction, stroke, orcomplications thereof. The vast majority of patients suffering acutecardiovascular events have prior exposure to at least one major riskfactor, such as cigarette smoking, abnormal blood lipid levels,hypertension, diabetes, abdominal obesity and low-grade inflammation.

Pathophysiologically, the major events of myocardial infarction andischemic stroke are caused by a sudden arrest of nutritive blood supplydue to a blood clot formation within the lumen of the arterial bloodvessel. In most cases, formation of the thrombus is precipitated byrupture of a vulnerable atherosclerotic plaque, which exposes chemicalagents that activate platelets and the plasma coagulation system. Theactivated platelets form a platelet plug that is armed bycoagulation-generated fibrin to form a blood clot that expands withinthe vessel lumen until it obstructs or blocks blood flow, which resultsin hypoxic tissue damage (so-called infarction). Thus, thromboticcardiovascular events occur as a result of two distinct processes, i.e.a slowly progressing long-term vascular atherosclerosis of the vesselwall, on the one hand, and a sudden acute clot formation that rapidlycauses flow arrest, on the other. Without wishing to be bound by theory,it is thought that the present invention solely relates to the latterprocess.

Recently, inflammation has been recognized as an important risk factorfor thrombotic events. Vascular inflammation is a characteristic featureof the atherosclerotic vessel wall, and inflammatory activity is astrong determinant of the susceptibility of the atherosclerotic plaqueto rupture and initiate intravascular clotting. Also, autoimmuneconditions with systemic inflammation, such as rheumatoid arthritis,systemic lupus erythematosus and different forms of vasculitides,markedly increase the risk of myocardial infarction and stroke.

Traditional approaches to prevent and treat cardiovascular events aretargeted: 1) to slow down the progression of the underlyingatherosclerotic process; 2) to prevent clot formation in case of aplaque rupture; or 3) to direct removal of an acute thrombotic flowobstruction. In short, antiatherosclerotic treatment aims at modulatingthe impact of general risk factors and includes dietary recommendations,weight loss, physical exercise, smoking cessation, cholesterol- andblood pressure treatment etc.

Prevention of clot formation mainly relies on the use of antiplateletdrugs that inhibit platelet activation and/or aggregation, but also insome cases includes thromboembolic prevention with oral anticoagulantssuch as warfarin. Post hoc treatment of acute atherothrombotic eventsrequires either direct pharmacological lysis of the clot by thrombolyticagents such as recombinant tissue-type plasminogen activator orpercutaneous mechanical dilation of the obstructed vessel.

Despite the fact that multiple-target anti-atherosclerotic therapy andclot prevention by antiplatelet agents have lowered the incidence ofmyocardial infarction and ischemic. stroke, such events still remain amajor population health problem. This shows that in patients withcardiovascular risk factors these prophylactic measures are insufficientto completely prevent the occurrence of atherothrombotic events.

Likewise, thrombotic conditions on the venous side of the circulation,as well as embolic complications thereof such as pulmonary embolism,still cause substantial morbidity and mortality. Venous thrombosis has adifferent clinical presentation and the relative importance of plateletactivation versus plasma coagulation are somewhat different, with apreponderance for the latter in venous thrombosis. However, despitethese differences, the major underlying mechanisms that cause thromboticvessel occlusions are similar to those operating on the arterialcirculation. Moreover, although unrelated to atherosclerosis as such,the risk of venous thrombosis is related to general cardiovascular riskfactors, such as inflammation and metabolic aberrations.

Taken together, existing therapy and general risk factor managementoffers insufficient protection against thrombotic events, both in thearterial and venous circulations, and cannot reverse the severeconsequences of such events. This creates a need for development ofnovel preventive and therapeutic targets, especially more effectiveapproaches that could prevent hazardous tissue ischemia, and ideally atsuch an early stage that symptoms have not yet occurred.

Interestingly, it has been found that, in an otherwise healthyindividual, there is a natural “last line of defense” system, which canbe activated if a clotting process, despite preventive measures, shouldoccur in the vasculature. In brief, initiation of a thrombotic mechanismboth on the arterial and venous sides of the circulation leads toactivation of the innermost cell layer of the blood vessel (theendothelium), and as a response the cells rapidly release large amountsof the clot-dissolving substance tissue-type plasminogen activator(t-PA). This raises luminal t-PA levels to similar levels as withclinical thrombolytic therapy (i.e. administration of recombinant t-PA),but the potency of this endogenous response is 100-fold greater due tothe extremely rapid onset of action.

Accumulating clinical, epidemiologic, and experimental data support thenotion that if this thromboprotective function of the blood vessel wallis intact, it offers a powerful defense against formation offlow-arresting thrombi. Unfortunately, however, the capacity for acutet-PA release is impaired in several conditions with increasedsusceptibility to thrombotic events. These include atherosclerosis,hypertension, abdominal obesity, smoking, sedentary lifestyle, andlow-grade inflammation. This impairment is most likely due to adiminished synthesis and thereby reduced availability of thefibrinolytic activator in the endothelial cells.

In addition, we and others have shown that the efficiency of theendogenous fibrinolytic response is reduced in patients with increasedrisk for an atherothrombotic event, such as in atherosclerosis(Osterlund, B., et al. Acta Anaesthesiol Scand 52, 1375-1384 (2008),Newby, D. E., et al. Circulation 103, 1936-1941 (2001)). Recent datasuggest that inflammation may be an underlying pathogenetic mechanismbehind the suppressed t-PA production in this state. We have shown thatprolonged exposure to the inflammatory cytokines tumor necrosis factoralpha (TNF-alpha) and interleukin-1 beta (IL-1b) causes a markedsuppression of the transcription of t-PA (Ulfhammer, E., et al. Journalof Thrombosis and Haemostasis 4, 1781-1789 (2006), Larsson, P., et al.Thromb Res 123, 342-351 (2008)). Interestingly, it is known that theatherosclerotic plaque is associated with a local, potentially severe,inflammatory activation in the vessel wall and it is conceivable thatthis inflammatory milieu hampers the fibrinolytic response in thespecific areas of the vasculature where it is pivotal to retain a highfibrinolytic capacity, thus increasing the risk of thrombotic events.Similarly, it is also likely that the increased incidence of thromboticevents in patients with systemic inflammatory conditions (e.g.autoimmune diseases and the metabolic syndrome), could also be relatedto a suppressive effect of circulating pro-inflammatory cytokines ont-PA synthesis and/or increased levels of plasminogen activatorinhibitor 1 (PAI-1).

Against this background, an alternative fourth approach to reduce theincidence of clinical thrombotic events should be to restore thecapacity of the fibrinolytic ‘last line of defense’ system in patientswith an impairment of its function. Extensive efforts have been made tofind a feasible means for enhancing basal as well as stimulatedendogenous fibrinolysis in subjects with a risk factor-associatedreduction of fibrinolytic capacity. However, previous attempts toameliorate t-PA synthesis with e.g. statins and retinoic acid have beendisappointing. Other means of increasing fibrinolysis by blockingnaturally occurring inhibitors of t-PA activity such as plasminogenactivator inhibitor-1 (PAI-1) and carboxypeptidase U (CPU) have alsobeen unsuccessful mainly due to limited drugability, such as poorpharmacokinetic properties of the drug candidates. The fibrinolyticactivity of t-PA is inhibited by plasminogen activator inhibitor 1(PAI-1) through complex-binding to the t-PA molecule. By virtue of itsantifibrinolytic effect, PAI-1 diminishes the ability to dissolve bloodclots and thereby increase the risk of clinical thrombotic events(Hrafnklsdottir et al, J Thromb Haemost 2004; 2:1960-8).

PAI-1 circulates in low concentrations in plasma (typically around 5-10ng/mL in morning samples), but in the population plasma PAI-1concentration shows a marked right-wardly skewed distribution.Generally, circulating PAI-1 levels increase with age. Elevated PAI-1levels predispose for thrombotic events. On an individual scale, levelsabove 100 ng/mL are considered to constitute a significant risk factorfor cardiovascular events, even in the absence of other traditional riskfactors. Moreover, elevated PAI-1 levels are frequently found inpatients with obesity-related metabolic disorders such as Type-2diabetes mellitus and the metabolic syndrome.

Circulating levels of PAI-1 show a pronounced circadian variation, withpeak levels around 06:00 hours and a trough around 16:00 hours asillustrated in FIG. 1 (see also e.g. Scheer and Shea, Blood 2014). Asexpected, the morning PAI-1 rise coincides with the temporal peakincidence for thrombotic events, such as myocardial infarction.

Patients with obesity and/or the metabolic syndrome have highercirculating PAI-1 levels and augmented circadian peaks as illustrated inFIG. 1. Plasma concentrations typically range between 15-60 ng/mL inmorning samples in these patients, but levels are non-normallydistributed with a pronounced positive skewness. Plasma PAI-1 levelsbetween 100-500 mg/mL in morning samples are not infrequently observedin obese patients with the metabolic syndrome. Thus, patients withobesity and/or the metabolic syndrome are at particular risk ofsuffering thrombotic events resulting from the inhibitory effect ofPAI-1 on the action of t-PA.

Therefore, it would be interesting to prevent cardiovascular events bylowering PAI-1, and more specifically to abrogate the early morning risein its plasma concentration. This approach would theoretically be evenmore efficient in patients with obesity and/or the metabolic syndrome.

We have now surprisingly found that valproic acid (VPA) potently reducesplasma PAI-1 levels, with such reduction, and corresponding reduction inPAI-1 activity, allowing for an increase in the activity of endogenoust-PA. Thus, administration of VPA in low doses in a manner such thatplasma levels of VPA, or metabolites thereof, coincide with peak plasmalevels of PAI-1 allows for an advantageous effect in the treatment orprevention of pathological conditions associated with excess fibrindeposition and/or thrombus formation.

WO 2012/120262 discusses the use of valproic acid in improving ornormalizing endogenous fibrinolysis impaired by local or systemicinflammation. However, it provides no suggestion that VPA may inhibitthe action of PAI-1 and, therefore, does not suggest the administrationof VPA to counteract (i.e. reduce) peak levels of PAI-1, thus providinga treatment (i.e. an improved treatment) for pathological conditionsassociated with excess fibrin deposition and/or thrombus formation.

US2007/0232528A1 describes controlled release formulations comprisingvalproic acid for use in the treatment of disorders such as cancer.These disclosures do not suggest the administration of WA to counteractpeak levels of PAI-1, for the treatment for pathological conditionsassociated with excess fibrin deposition and/or thrombus formation, andso do not suggest formulations designed for this use.

DESCRIPTION OF THE INVENTION

The present invention relates to fibrin degradation or breakdown (alsocalled fibrinolysis), and more particularly compositions and methods forthe treatment of pathological conditions associated with excess fibrindeposition and/or thrombus formation (e.g. thrombus formation).

In particular, the present invention relates to methods of usingvalproic acid, or pharmaceutically acceptable salts thereof, in thetreatment or prevention of pathological conditions associated withexcess fibrin deposition and/or thrombus formation.

The present invention also provides pharmaceutical compositionsformulated to delay the release of valproic acid, or pharmaceuticallyacceptable salts thereof, in a manner suitable for use in such methods.

Medical Treatments

As described herein, it has been found that valproic acid, orpharmaceutically acceptable salts thereof, is able to inhibit theactivity of PAI-1 (e.g. through reduction of PAI-1 levels), which itselfis an inhibitor of t-PA. As a consequence, valproic acid, orpharmaceutically acceptable salts thereof, is able to increase theeffects of t-PA and, therefore, is of use in the treatment or preventionof pathological conditions associated with excess fibrin depositionand/or thrombus formation.

In particular, the inventors have unexpectedly found that human subjectstreated with VPA had reduced circulating levels of PAI-1. In healthy mencirculating plasma levels of PAI-1 were significantly reduced by morethan 50% after VPA treatment and in patients with coronaryatherosclerosis by about 45%, which results are further described inExample 1 as provided herein.

The finding that VPA treatment lowers plasma levels of PAI-1 in man wasunexpected given that in vitro data from cultured endothelial cells (oneof the believed producers of plasma PAI-1) did not show a decrease ofPAI-1 mRNA levels after VPA treatment, rather a slight but significant30% increase in PAI-1 production. These studies also did not detect anyeffects of VPA on plasma PAI-1 in the in vivo models in pig (Svennerholmet al., PLoS One. 2014 May 12; 9(5):e97260. doi:10.1371/journal.pone.0097260. eCollection 2014) or in mouse (unpublisheddata).

In a first aspect of the invention, there is provided valproic acid, ora pharmaceutically acceptable salt thereof, for use in treating orpreventing a pathological condition associated with excess fibrindeposition and/or thrombus formation, wherein said treatment comprisesadministering at least one dose of valproic acid, or a pharmaceuticallyacceptable salt thereof, to a patient such that the maximum plasmaconcentration (Cmax) of valproic acid, or a salt and/or metabolitethereof, in the patient occurs during a time period that is from fourhours before to one hour after the maximum plasma concentration (Cmax)of PAI-1 in the patient.

In an alternative first aspect of the invention, there is provided theuse of valproic acid, or a pharmaceutically acceptable salt thereof, inthe manufacture of a medicament for use in treating or preventing apathological condition associated with excess fibrin deposition and/orthrombus formation, wherein said treatment comprises administering atleast one dose of valproic acid, or a pharmaceutically acceptable saltthereof, to a patient such that the maximum plasma concentration (Cmax)of valproic acid, or a salt and/or metabolite thereof, in the patientoccurs during a time period that is from four hours before to one hourafter the maximum plasma concentration (Cmax) of PAI-1 in the patient.

In a further alternative first aspect of the invention, there is amethod of treating or preventing a pathological condition associatedwith excess fibrin deposition and/or thrombus formation in a patient inneed thereof comprising administering at least one dose of atherapeutically effective amount of valproic acid, or a pharmaceuticallyacceptable salt thereof, to a patient such that the maximum plasmaconcentration (Cmax) of valproic acid, or a salt and/or metabolitethereof, in the patient occurs during a time period that is from fourhours before to one hour after the maximum plasma concentration (Cmax)of PAI-1 in the patient.

The skilled person will understand that references herein to embodimentsof particular aspects of the invention will include references to allother embodiments of those aspects of the invention. As such, any moreor more embodiments of any aspect of the invention may be combined withany one or more other such embodiments in order to form more particularembodiments without departing from the disclosure of the invention asprovided herein.

As used herein, references to a pathological condition associated withexcess fibrin deposition and/or thrombus formation will refer inparticular to pathological conditions associated with thrombusformation.

In a particular embodiment of the first aspect of the invention, themaximum plasma concentration (Cmax) of valproic acid, or a salt and/ormetabolite thereof, in the patient occurs during a time period that isfrom four hours before to the time of the maximum plasma concentration(Cmax) of PAI-1 in the patient.

In another particular embodiment of the first aspect of the invention,the maximum plasma concentration (Cmax) of valproic acid, or a saltand/or metabolite thereof, in the patient occurs during a time periodthat is from three hours before (e.g. two hours before) to one hourafter the maximum plasma concentration (Cmax) of PAI-1 in the patient.

In a more particular embodiment of the first aspect of the invention,the maximum plasma concentration (Cmax) of valproic acid, or a saltand/or metabolite thereof, in the patient occurs during a time periodthat is from three hours before (e.g. two hours before) to the time ofthe maximum plasma concentration (Cmax) of PAI-1 in the patient.

In a second aspect of the invention, there is provided valproic acid, ora pharmaceutically acceptable salt thereof, for use in treating orpreventing a pathological condition associated with excess fibrindeposition and/or thrombus formation, wherein said treatment comprisesadministering at least one dose of Valproic acid, or a pharmaceuticallyacceptable salt thereof, to a patient such that at the time when thepatient experiences the maximum plasma concentration (Cmax) of PAI-1,the patient has a plasma concentration of valproic acid, or a saltand/or metabolite thereof, that is at least about 10 to about 100 μg/ml,such as at least about 10 (e.g. at least about 20, about 30, about 40,about 50, about 60, about 70, about 80, about 90 or about 100 μg/ml).

In an alternative second aspect of the invention, there is provided theuse of valproic acid, or a pharmaceutically acceptable salt thereof, inthe manufacture of a medicament for use in treating or preventing apathological condition associated with excess fibrin deposition and/orthrombus formation, wherein said treatment comprises administering atleast one dose of Valproic acid, or a pharmaceutically acceptable saltthereof, to a patient such that at the time when the patient experiencesthe maximum plasma concentration (Cmax) of PAI-1, the patient has aplasma concentration of valproic acid, or a salt and/or metabolitethereof, that is at least about 10 to about 100 μg/ml, such as e.g. atleast about 10, about 20, about 30, about 40, about 50, about 60, about70, about 80, about 90 or about 100 μg/ml).

In a further alternative second aspect of the invention, there isprovided a method of treating or preventing a pathological conditionassociated with excess fibrin deposition in a patient in need thereofcomprising administering at least one therapeutically effective dose ofvalproic acid, or a pharmaceutically acceptable salt thereof, to apatient such that at the time when the patient experiences the maximumplasma concentration (Cmax) of PAI-1, the patient has a plasmaconcentration of valproic acid, or a salt and/or metabolite thereof,that is at least about 10 to about 100 μg/ml, such as e.g. at leastabout 10, about 20, about 30, about 40, about 50, about 60, about 70,about 80, about 90 or about 100 μg/ml).

For the avoidance of doubt, the skilled person will understand thatreferences to compounds of the Invention as provided herein above willapply to the second aspect of the invention (and alternative aspectsand/or particular embodiments thereof) in the same manner as they applyto all other aspects of the invention described herein (and alternativeaspects and/or particular embodiments thereof).

As used herein, the term therapeutic window will be understood to referto plasma levels of the relevant compound, or a salt and/or metabolitethereof, at which the relevant (i.e. normally associated) therapeuticeffect of that compound will typically be observed. The term may referto a range of plasma levels or to a specific plasma level.

As used herein, the reference to an amount per milliliter (/ml) will beunderstood to refer to an amount per milliliter of plasma (i.e. bloodplasma of the patient). As used herein, the reference to molarconcentration will be understood to refer to a concentration in plasma(i.e. blood plasma of the patient).

In alternative second aspects of the invention, the patient has a plasmaconcentration of valproic acid, or a salt and/or metabolite thereof,that is below about 50 to about 170 μg/ml (such as e.g. below about 50,about 70, about 90, about 110, about 130, about 150, or about 170μg/ml).

In further alternative second aspects of the invention, the patient hasa plasma concentration of valproic acid, or a salt and/or metabolitethereof, that is at least about 70 to about 700 μM (such as e.g. atleast about 70, about 140, about 210, about 280, about 350, about 420,about 490, about 560, about 630 or about 700 μM).

In yet further alternative second aspects of the invention, the patienthas a plasma concentration of valproic acid, or a salt and/or metabolitethereof, that is below about 350 to about 1200 μM (such as e.g. belowabout 350, about 490, about 630, about 770, about 910, about 1050, orabout 1190 μM).

For the avoidance of doubt, the skilled person will understand thatreferences to certain maximum amounts and concentrations in plasma inthe second aspect of the invention may also require a minimum of atherapeutically effective amount in said plasma.

In particular, the skilled person will understand that references tocertain maximum (i.e. where values are indicated as being “below”) andminimum (i.e. where values are indicated as being “at least”) amountand/or concentrations in plasma may be combined to form ranges (i.e.wherein the amount in plasma is in a range that is from the minimumvalue to the maximum value).

For example, in one embodiment of the second aspect of the invention,the patient has a plasma concentration of valproic acid, or a saltand/or metabolite thereof, that is about 10 to about 170 μg/ml.

In other such embodiments, the patient has a plasma concentration ofvalproic acid, or a salt and/or metabolite thereof, that is:

from about 10 to about 70 ug/ml (or from about 50 to about 90, about 70to about 110, about 90 to about 130, about 110 to about 150, about 130to about 170, or about 150 to about 190 ug/ml);

from about 10 to about 50 ug/ml (or from about 10 and to about 100,about 30 to about, about 50 to about 170, or about 70 to about 190ug/ml); from about 30 to about 190 ug/ml (e.g. about 50 to about 170,about 70 to about 150, about 90 to about 130, about 30 to about 110,about 50 to about 130, or about 70 to about 170 ug/ml).

The skilled person will understand that references to certain minimumplasma levels herein (e.g. in the second aspect of the invention) willinclude references to such levels at a time when the patient has reacheda steady state of valproic acid, or a salt and/or metabolite thereof, inplasma. Moreover, the skilled person will understand that references tothe patient reaching a steady state may refer to the plasma levelsachieved after said patient has been treated with compounds of theinvention (at a therapeutically-effective dose thereof) for at least twoto five days (e.g. at least five days).

The skilled person will also understand that the references to maximumand minimum plasma levels in the second aspect of the invention(including all embodiments and alternative aspects thereof) may alsoapply to the plasma levels observed for the Cmax of valproic acid, or asalt and/or metabolite thereof, as referred to in other aspects of theinvention (such as the first aspect of the invention).

In a third aspect of the invention, there is provided valproic acid, ora pharmaceutically acceptable salt thereof, for use in treating orpreventing a pathological condition associated with excess fibrindeposition and/or thrombus formation, wherein said treatment comprisesadministering a dose of valproic acid, or a pharmaceutically acceptablesalt thereof, to a patient during a time period from about 20:00 hoursto about 06:00 hours.

In an alternative third aspect of the invention, there is provided theuse of valproic acid, or a pharmaceutically acceptable salt thereof, inthe manufacture of a medicament for use in treating or preventing apathological condition associated with excess fibrin deposition and/orthrombus formation, wherein said treatment comprises administering adose of valproic acid, or a pharmaceutically acceptable salt thereof, toa patient during a time period from about 20:00 hours to about 06:00hours.

In a further alternative third aspect of the invention, there isprovided a method of treating or preventing a pathological conditionassociated with excess fibrin deposition and/or thrombus formation in apatient in need thereof comprising administering a therapeuticallyeffective dose of valproic acid, or a pharmaceutically acceptable saltthereof, to a patient during a time period from about 20:00 hours toabout 06:00 hours.

In a particular embodiment of the third aspect of the invention, thetreatment comprises administering a therapeutically effective dose ofvalproic acid, or a pharmaceutically acceptable salt thereof, to apatient during a time period from about 21:00 hours to about 05:00 hours(e.g. about 22:00 hours to about 04:00 hours).

The skilled person will understand that timings referred to using the24-hour system may also be referred to as timings using the 12-hoursystem (i.e. with AM and PM denoting times before and after 12:00 noon,respectively). For example, 20:00 may also be referred to as 8:00 PM,and 06:00 as 6:00 AM.

In a particular embodiment of the third aspect of the invention(particularly wherein the treatment is administered as a pharmaceuticalcomposition that is not formulated for delayed release of the activeingredient), the treatment comprises administering a therapeuticallyeffective dose of valproic acid, or a pharmaceutically acceptable saltthereof, to a patient during a time period from about 02:00 hours toabout 06:00 hours (e.g. about 03:00 hours to about 05:00 hours, such asabout 04:00 hours).

In another particular embodiment of the third aspect of the invention(particularly wherein the treatment is administered as a pharmaceuticalcomposition that is formulated for delayed release of the activeingredient, such as those described in the eight aspect of the inventionherein), the treatment comprises administering a therapeuticallyeffective dose of valproic acid, or a pharmaceutically acceptable saltthereof, to a patient during a time period from about 20:00 hours toabout 00:00 hours (e.g. about 21:00 hours to about 23:00 hours, such asat about 22:00 hours).

In alternative embodiments of the third aspect of the invention, thetreatment comprises administering a therapeutically effective dose ofvalproic acid, or a pharmaceutically acceptable salt thereof, to apatient during a time period determined based on the release profile ofthat formulation in order to provide a plasma concentration of valproicacid, or a salt and/or metabolite thereof, as required in the firstand/or second aspect of the invention.

As described herein, the skilled person will be able to determine how toadminister compounds of the invention in a manner (e.g. during a certaintime period) in order to achieve parameters described herein (such asthose described in the first and second aspects of the invention).

For the avoidance of doubt, in particular embodiments of the thirdaspect of the invention, the dose referred to is a single dose, whichwill indicate that the dose is the only dose of the compound given tothe patient during a (e.g. the relevant) 24 hour period.

In a fourth aspect of invention, there is provided valproic acid, or apharmaceutically acceptable salt thereof, for use in treating orpreventing a pathological condition associated with excess fibrindeposition and/or thrombus formation, wherein said treatment comprisesadministering a pharmaceutical composition comprising a dose of valproicacid, or a pharmaceutically acceptable salt thereof, to a patient at atime and in a form such that substantially all of the valproic acid, ora pharmaceutically acceptable salt thereof, is released from thecomposition during a time period from about 02:00 hours to about 06:00hours.

In an alternative fourth aspect of invention, there is provided the useof valproic acid, or a pharmaceutically acceptable salt thereof, in themanufacture of a medicament for use in treating or preventing apathological condition associated with excess fibrin deposition and/orthrombus formation, wherein said treatment comprises administering apharmaceutical composition comprising a dose of valproic acid, or apharmaceutically acceptable salt thereof, to a patient at a time and ina form such that substantially all of the valproic acid, or apharmaceutically acceptable salt thereof, is released from thecomposition during a time period from about 02:00 hours to about 06:00hours.

In a further alternative fourth aspect of invention, there is provided amethod of treating or preventing a pathological condition associatedwith excess fibrin deposition and/or thrombus formation in a patient inneed thereof comprising administering a pharmaceutical compositioncomprising a therapeutically effective dose of valproic acid, or apharmaceutically acceptable salt thereof, to a patient at a time and ina form such that substantially all of the valproic acid, or apharmaceutically acceptable salt thereof, is released from thecomposition during a time period from about 02:00 hours to about 06:00hours.

In a particular embodiment of the fourth aspect of the invention, thetreatment comprises administering a pharmaceutical compositioncomprising a therapeutically effective dose of valproic acid, or apharmaceutically acceptable salt thereof, to a patient at a time and ina form such that substantially all of the valproic acid, or apharmaceutically acceptable salt thereof, is released from thecomposition during a time period from about 03:00 hours to about 05:00hours (e.g. from about 04:00 hours to about 05:00 hours, such as atabout 05:00 hours).

In a particular embodiment of the fourth aspect of the invention, thetreatment comprises administering a pharmaceutical composition asdescribed in the eight aspect of the invention herein below (includingall embodiments thereof).

In a fifth aspect of the invention, there is provided valproic acid, ora pharmaceutically acceptable salt thereof, for use in treating orpreventing a pathological condition associated with excess fibrindeposition and/or thrombus formation in a patient, wherein saidtreatment comprises:

-   -   (i) monitoring the plasma concentration of PAI-1 in the patient        in order to determine the time at, or time period during which,        the maximum plasma concentration of PAI-1 occurs;    -   (ii) administering at least one dose of valproic acid, or a        pharmaceutically acceptable salt thereof, to the patient such        that the maximum plasma concentration (Cmax) of valproic acid,        or a salt and/or metabolite thereof, in the patient occurs        during a time period that is from four hours before to one hour        after the time at which, or time period during which, the        maximum plasma concentration of PAI-1 occurs.

In an alternative fifth aspect of the invention, there is provided theuse of valproic acid, or a pharmaceutically acceptable salt thereof, inthe manufacture of a medicament for use in treating or preventing apathological condition associated with excess fibrin deposition and/orthrombus formation in a patient, wherein said treatment comprises:

-   -   (i) monitoring the plasma concentration of PAI-1 in the patient        in order to determine the time at, or time period during which,        the maximum plasma concentration of PAI-1 occurs; and    -   (ii) administering at least one dose of valproic acid, or a        pharmaceutically acceptable salt thereof, to the patient such        that the maximum plasma concentration (Cmax) of valproic acid,        or a salt and/or metabolite thereof, in the patient occurs        during a time period that is from four hours before to one hour        after the time at which, or time period during which, the        maximum plasma concentration of PAI-1 occurs.

In a further alternative fifth aspect of the invention, there isprovided a method of treating or preventing a pathological conditionassociated with excess fibrin deposition and/or thrombus formation in apatient in need thereof comprising the steps of:

-   -   (i) monitoring the plasma concentration of PAI-1 in the patient        in order to determine the time at, or time period during which,        the maximum plasma concentration of PAI-1 occurs; and    -   (ii) administering at least one therapeutically effective dose        of valproic acid, or a pharmaceutically acceptable salt thereof,        to the patient such that the maximum plasma concentration (Cmax)        of valproic acid, or a salt and/or metabolite thereof, in the        patient occurs during a time period that is from four hours        before to one hour after the time at which, or time period        during which, the maximum plasma concentration of PAI-1 occurs.

As described herein, plasma concentrations of PAI-1 may be monitoredusing techniques well-known to those skilled in the art. For instance,PAI-1 levels are generally measured in plasma. Blood may be collectedfrom an antecubital syringe regularly e.g. every hour, every second houror every third hour throughout 24 hours. The blood samples areimmediately centrifuged to separate plasma from the serum. ThereafterPAI-1 levels in plasma are determined by using commercially availableELISA-kits, such as Coaliza® PAI-1 (Chromogenix), TriniLIZE® PAI-1(Trinity Biotech), Imubind® Plasma PAI-1 (American Diagnostica),Zymutest PAI-1 (Hyphen Biomed), Milliplex PAI-1 (MerckMillipore), NovexPAI-1 human Elisa kit (Life technology), PAI1 (SERPINE1) Human ELISA Kit(Abcam, ab108891).

In a particular embodiment of the fifth aspect of the invention, themaximum plasma concentration (Cmax) of valproic acid, or a salt and/ormetabolite thereof, in the patient occurs during a time period that isfrom four hours before (e.g. three hours before, such as 2 hours beforeor 1 hour before or 0.5 hours before) to the time of the maximum plasmaconcentration (Cmax) of PAI-1 in the patient.

In another particular embodiment of the fifth aspect of the invention,the maximum plasma concentration (Cmax) of valproic acid, or a saltand/or metabolite thereof, in the patient occurs during a time periodthat is from three hours before (e.g. two hours before) to one hourafter the maximum plasma concentration (Cmax) of PAI-1 in the patient.

In a more particular embodiment of the fifth aspect of the invention,the maximum plasma concentration (Cmax) of valproic acid, or a saltand/or metabolite thereof, in the patient occurs during a time periodthat is from three hours before (e.g. two hours before) to the time ofthe maximum plasma concentration (Cmax) of PAI-1 in the patient.

The skilled person will understand that the timing and level of the Cmaxof VPA will depend on the dose administered (and, to some extent, theform in which that dose is administered). The skilled person will beable to measure the plasma concentration of VPA, or a metabolite and/orsalt thereof, and determine the timing and level of the Cmax (and, ifnecessary, to adjust the dose and form of VPA accordingly). Particulardoses (i.e. therapeutic doses) of VPA that may be administered and Cmaxlevels that may be obtained include those as described herein.

In a sixth aspect of the invention, there is provided valproic acid, ora pharmaceutically acceptable salt thereof, for use in treating orpreventing a pathological condition associated with excess fibrindeposition and/or thrombus formation in a patient, wherein saidtreatment comprises:

-   -   (i) monitoring the plasma concentration of PAI-1 in the patient        in order to determine the time at, or time period during which,        the maximum plasma concentration of PAI-1 occurs; and    -   (ii) administering at least one dose of valproic acid, or a        pharmaceutically acceptable salt thereof, to the patient such        that at the time when the patient experiences the maximum plasma        concentration of PAI-1, the patient has a plasma concentration        of valproic acid, or a salt and/or metabolite thereof, that is        at least about 10 to about 100 μg/ml (such as e.g. at least        about 10, about 20, about 30, about 40, about 50, about 60,        about 70, about 80, about 90 or about 100 μg/ml).

In an alternative sixth aspect of the invention, there is provided theuse of valproic acid, or a pharmaceutically acceptable salt thereof, inthe manufacture of a medicament for use in treating or preventing apathological condition associated with excess fibrin deposition and/orthrombus formation in a patient, wherein said treatment comprises:

-   -   (i) monitoring the plasma concentration of PAI-1 in the patient        in order to determine the time at, or time period during which,        the maximum plasma concentration of PAI-1 occurs; and    -   (ii) administering at least one dose of valproic acid, or a        pharmaceutically acceptable salt thereof, to the patient such        that at the time when the patient experiences the maximum plasma        concentration of PAI-1, the patient has a plasma concentration        of valproic acid, or a salt and/or metabolite thereof, that is        at least about 10 to about 100 μg/ml (such as e.g. at least        about 10, about 20, about 30, about 40, about 50, about 60,        about 70, about 80, about 90 or about 100 μg/ml).

In a further alternative sixth aspect of the invention, there isprovided a method of treating or preventing a pathological conditionassociated with excess fibrin deposition and/or thrombus formation in apatient in need thereof comprising the steps of:

-   -   (i) monitoring the plasma concentration of PAI-1 in the patient        in order to determine the time at, or time period during which,        the maximum plasma concentration of PAI-1 occurs; and    -   (ii) administering at least one therapeutically effective dose        of valproic acid, or a pharmaceutically acceptable salt thereof,        to the patient such that at the time when the patient        experiences the maximum plasma concentration of PAI-1, the        patient has a plasma concentration of valproic acid, or a salt        and/or metabolite thereof, that is at least about 10 to about        100 μg/ml (such as e.g. at least about 10, about 20, about 30,        about 40, about 50, about 60, about 70, about 80, about 90 or        about 100 μg/ml).

In alternative sixth aspects of the invention, the patient has a plasmaconcentration of valproic acid, or a salt and/or metabolite thereof,that is below about 50 to about 170 μg/ml (such as e.g. below about 50,about 70, about 90, about 110, about 130, about 150, or about 170μg/ml).

In further alternative sixth aspects of the invention, the patient has aplasma concentration of valproic acid, or a salt and/or metabolitethereof, that is at least about 70 to about 700 μM (such as e.g. atleast about 70, about 140, about 210, about 280, about 350, about 420,about 490, about 560, about 630 or about 700 μM).

In yet further alternative sixth aspects of the invention, the patienthas a plasma concentration of valproic acid, or a salt and/or metabolitethereof, that is below about 350 to about 1200 μM (such as e.g. belowabout 350, about 490, about 630, about 770, about 910, about 1050, orabout 1190 μM).

For the avoidance of doubt, the skilled person will understand thatreferences herein to levels and concentrations (e.g. plasma levels andplasma concentrations) of “valproic acid, or a salt and/or metabolitethereof” will refer in particular to levels and concentrations (e.g.plasma levels and plasma concentrations) of valproic acid.

Again, the skilled person will understand that references to certainmaximum amounts and concentrations in plasma in the sixth aspect of theinvention may also require a minimum of a therapeutically effectiveamount in said plasma. Moreover, the skilled person will understand thatreferences to certain maximum (i.e. where values are indicated as being“below”) and minimum (i.e. where values are indicated as being “atleast”) amount and/or concentrations in plasma may be combined to formranges (i.e. wherein the amount in plasma is in a range that is from theminimum value to the maximum value).

For example, in one embodiment of the sixth aspect of the invention, thepatient has a plasma concentration of valproic acid, or a salt and/ormetabolite thereof, that is about 10 to about 170 ug/ml. In other suchembodiments, the patient has a plasma concentration of valproic acid, ora salt and/or metabolite thereof, that is:

from about 10 to about 70 ug/ml (or from about 50 to about 90, about 70to about 110, about 90 to about 130, about 110 to about 150, about 130to about 170, or about 150 to about 190 ug/ml);

from about 10 to about 50 ug/ml (e.g. from about 10 and to about 100,about 30 to about, about 50 to about 170, or about 70 to about 190ug/ml);

from about 30 to about 190 ug/ml (e.g. about 50 to about 170, about 70to about 150, about 90 to about 130, about 30 to about 110, about 50 toabout 130, or about 70 to about 170 ug/ml).

In a seventh aspect of the invention, there is provided valproic acid,or a pharmaceutically acceptable salt thereof, for use in treating orpreventing a pathological condition associated with excess fibrindeposition and/or thrombus formation, wherein said treatment comprisesadministering a single dose of valproic acid, or a pharmaceuticallyacceptable salt thereof, to a patient in a 24 hour period, wherein thedose is from about 50 mg to about 1000 mg (such as about 200 mg to about600 mg).

In an alternative seventh aspect of the invention, there is provided theuse of valproic acid, or a pharmaceutically acceptable salt thereof, inthe manufacture of a medicament for use in treating or preventing apathological condition associated with excess fibrin deposition and/orthrombus formation, wherein said treatment comprises administering asingle dose of valproic acid, or a pharmaceutically acceptable saltthereof, to a patient in a 24 hour period, wherein the dose is fromabout 50 mg to about 1000 mg (such as about 200 mg to about 600 mg).

In a further alternative seventh aspect of the invention, there isprovided a method of treating or preventing a pathological conditionassociated with excess fibrin deposition and/or thrombus formation in apatient in need thereof comprising administering a single,therapeutically effective dose of valproic acid, or a pharmaceuticallyacceptable salt thereof, to a patient in a 24 hour period, wherein thedose is from about 50 mg to about 1000 mg (such as about 200 mg to about600 mg).

Unless otherwise stated or apparent from the context (e.g. whendiscussed in reference to a specific formulation), references to thedose of compounds of the invention (e.g. the dose of valproic acid or apharmaceutically acceptable salt thereof) will be understood to refer tothe dose of valproic acid (i.e. the dose of valproic acid itself, or theeffective (i.e. equivalent) dose of valprioic acid when administered inthe form that includes or consists of one or more salt thereof.

In a particular embodiment of the seventh aspect of the invention, thedose is from about 200 mg to about 400 mg, such as about 400 or about300 mg. In another particular embodiment of the seventh aspect of theinvention, the dose is from about 300 mg to about 500 mg, such as about350 mg. In another particular embodiment of the seventh aspect of theinvention, the dose is from about 400 mg to about 600 mg, such as about450 or about 550 mg. In another particular embodiment of the seventhaspect of the invention, the dose is from about 400 mg to about 800 mg,such as about 575, about 650 or about 700 mg.

Again, for the avoidance of doubt, all references herein to particularaspects of the invention (e.g. the first aspect of the invention) willinclude references to all alternative such aspects of the invention(e.g. the alternative and further alternative first aspects of theinvention).

Moreover, the skilled person will understand that all embodiments,preferences, particular definitions and the like referred to herein maybe combined with any one or more other embodiments, preferences,particular definitions and the like also referred to herein.

When used herein in reference to a value or an amount (including anamount of time), the terms “about”, “around” and “approximately” will beunderstood as referring to a value that is within 10% of the valuedefined. When used herein in reference to a specific point in time(including the start or end of a period of time), the terms “about” and“around” will be understood as referring to a value that is within 30minutes (e.g. within 20 minutes, such as within 10 minutes) of thatspecific time. Further, it is contemplated that each reference to theterms “about”, “around” and “approximately” (e.g. in relation to timesand amounts) may be deleted throughout.

As used herein, the term “compounds of the invention” will refer tovalproic acid and pharmaceutically acceptable salts thereof. The skilledperson will understand that references to valproic acid andpharmaceutically acceptable salts thereof (e.g. references to “valproicacid, or a pharmaceutically acceptable salt thereof”) may includereferences to mixtures of different pharmaceutically acceptable salts,and references to mixtures of valproic acid (i.e. in non-salt form) andpharmaceutically acceptable salts thereof (including mixtures of suchsalts), all of which may be referred to as compounds of the invention.

As used herein, the skilled person will understand that references to“preventing” a particular condition may also be referred to as“prophylaxis” of said condition, and vice versa. Thus, each referenceherein to “preventing” a condition may be replaced with a reference to“prophylaxis” of said condition.

The skilled person will understand that the terms “treatment” and“treating” when used herein take their normal meanings in the field ofmedicine. In particular, these terms may refer to achieving a reductionin the severity of one or more clinical symptom associated with therelevant condition.

The skilled person will also understand that the terms “prevention” and“preventing” when used herein take their normal meanings in the field ofmedicine. In particular, these terms may refer to achieving a reductionin the likelihood of (the patient) developing the relevant condition(for example, a reduction of at least 10% when compared to the baselinelevel, such as a reduction of at least 20% or, more particularly, areduction of at least 30%).

The skilled person will also understand that references to prophylaxsis(or prevention) of a particular condition may also include the treatmentof another condition. For example, treatment of a primary condition mayalso be considered to be a form of prophylaxsis of a secondarycondition.

In particular embodiments of the first to seventh aspects of theinvention (including all alternative aspects), there are providedcompounds for use in (and/or uses in and/or methods for) preventing apathological condition associated with excess fibrin deposition and/orthrombus formation (particularly, thrombus formation).

As used herein, the term “pathological conditions” will be understood torefer to identifiable diseases or disorders.

As described herein, pathological conditions that may be treated orprevented in accordance with the invention associated with excess fibrindeposition and/or thrombus formation. These include, but are not limitedto, atherosclerosis, myocardial infarction, ischemic stroke, deep veinthrombosis, superficial vein thrombosis, thrombophlebitis, pulmonaryembolism, disseminated intravascular coagulation, renal vascular diseaseand intermittent claudication (e.g. atherosclerosis, myocardialinfarction, ischemic stroke, deep vein thrombosis, pulmonary embolism,disseminated intravascular coagulation, renal vascular disease andintermittent claudication).

Thus, in particular embodiments of the first to seventh aspects of theinvention, the pathological condition associated with excess fibrindeposition and/or thrombus formation is selected from the groupconsisting of atherosclerosis, myocardial infarction, ischemic stroke,deep vein thrombosis, pulmonary embolism, disseminated intravascularcoagulation, renal vascular disease and intermittent claudication.

Thus, in more particular embodiments of the first to seventh aspects ofthe invention, the pathological condition associated with excess fibrindeposition and/or thrombus formation is selected from the groupconsisting of myocardial infarction, ischemic stroke and pulmonaryembolism.

In other more particular embodiments of the first to seventh aspects ofthe invention, the pathological condition associated with excess fibrindeposition and/or thrombus formation is selected from the groupconsisting of myocardial infarction and ischemic stroke (such asmyocardial infarction).

The skilled person will understand that references to ischemic strokeinclude references to major stroke events (i.e. those caused byprolonged impairment of blood flow), minor strokes and transientischemic attacks (TIAs).

Thus, in more particular embodiments of the first to seventh aspects ofthe invention, the pathological condition associated with excess fibrindeposition and/or thrombus formation is ischemic stroke, such as a majorischemic stroke, minor ischemic stroke or a TIA.

In even more particular embodiments of the first to seventh aspects ofthe invention, the pathological condition associated with excess fibrindeposition and/or thrombus formation is ischemic stroke, such as a majorischemic stroke and minor ischemic stroke.

In particular, it is believed that compounds of the invention, whenadministered in accordance with the dosage regimes defined above (e.g.in the first to seventh aspects of the invention), may be of particularuse in preventing a pathological condition associated with excess fibrindeposition and/or thrombus formation (such as ischemic stroke and/ormyocardial infarction). Thus, all references to treating and preventingsuch conditions herein will include particular references to preventingsuch conditions.

Thus, in yet more particular embodiments of the first to seventh aspectsof the invention, treating or preventing a pathological conditionassociated with excess fibrin deposition and/or thrombus formation willrefer to preventing ischemic stroke, such as a major ischemic stroke,minor ischemic stroke or a TIA.

As discussed above, thrombotic cardiovascular events occur as a resultof two distinct processes, i.e. a slowly progressing long-term vascularatherosclerosis of the vessel wall, on the one hand, and a sudden acuteclot formation that rapidly causes flow arrest, on the other. Particularpathological conditions that may be treated are those relating to thelatter process.

In particular embodiments of the first to seventh aspects of theinvention, pathological conditions that may be treated or prevented inaccordance with the invention are those that are caused wholly or atleast in part by an increased fibrin deposition and/or reducedfibrinolytic capacity due to local or systemic inflammation. Theseinclude, but are not limited to, myocardial infarction, stable anginapectoris, unstable angina pectoris, intermittent claudication, ischemicstroke, transient ischemic attack, deep vein thrombosis and pulmonaryembolism. These conditions may display elevated PAI-1 levels in plasma.

In particular embodiments of the first to seventh aspects of theinvention, the pathological condition may be selected from the groupconsisting of deep vein thrombosis and pulmonary embolism.

In particular embodiments of the first to seventh aspects of theinvention, the pathological condition is deep vein thrombosis.

In particular embodiments of the first to seventh aspects of theinvention, the pathological condition may be selected from the groupconsisting of superficial vein thrombosis and thrombophlebitis.

In more particular embodiments of the first to seventh aspects of theinvention, the pathological condition is superficial vein thrombosis.

In more particular embodiments of the first to seventh aspects of theinvention, the pathological condition is thrombophlebitis.

In addition, pathological conditions that can be treated in accordancewith the invention are those that are caused wholly or at least in partby an increased fibrin deposition and/or reduced fibrinolytic capacitydue to local or systemic inflammation. These include but are not limitedto atherosclerosis, the metabolic syndrome, diabetes, disseminatedintravascular coagulation, rheumatoid arthritis, glomerulo-nephritis,systematic lupus erythematosis, vasculitides, autoimmune neuropathies,and granulomatous disease as well as inflammation associated with otherconditions (such as the metabolic syndrome, diabetes, disseminatedintravascular coagulation, rheumatoid arthritis, glomerulo-nephritis,systematic lupus erythematosis, vasculitides, autoimmune neuropathies,and granulomatous disease as well as inflammation associated with otherconditions).

In addition to traditional diagnosis of a systemic or local inflammationby a physician as is known in the art, a local or systemic inflammationcan be determined in patients using one or more biomarkers coupled toinflammation. These biomarkers include, but are not limited to, Creactive protein, TNF-alpha, high sensitive C-reactive protein (hs-CRP),fibrinogen, IL-1beta, and IL-6. Particular methods for determiningwhether a patient has systemic or local inflammation include thosedescribed hereinafter.

In addition, atherosclerotic plaques are known to be associated with avery localized inflammatory process. Hence, local inflammation may alsobe indirectly determined by the presence of atherosclerotic plaques asdiagnosed by vascular ultrasound or other imaging techniques.

The skilled person will understand that, to identify a poor level offibrinolysis in a patient (i.e. reduced fibrinolytic capacity), thereare a few different alternatives available. For example, highcirculating levels of PAI-1 are generally considered to be indicative ofpoor fibrinolysis, and this can be measured in plasma by commerciallyavailable methods (including but not limited by Coaliza® PAI-1(Chromgenix), TriniLIZE® PAI-1 (Trinity Biotech), Imubind® Plasma PAI-1(American Diagnostica), Zymutest PAI-1 (Hyphen Biomed), Milliplex PAI-1(MerckMillipore), Novex PAI-1 human Elisa kit (Life technology), PAI1(SERPINEI) Human ELISA Kit (Abcam, ab108891)). Further, low systemiclevels of free, active t-PA is also an indicator of general poorfibrinolysis and can also be measured by commercial methods (TriniLIZE®t-PA antigen and activity (Trinity Biotech), as is the presence of alow-producer (T) genotype of the t-PA-7351 C/T polymorphism. Functionalassays measuring clot lysis time have also been used to assess globalfibrinolysis (Thrombinoscope™ (Synapse, BV, Maastricht, theNetherlands), IL/ROTEM® (Term International GmbH, Munich, Germany), TEG®(Haemoscope, Niles), CloFAL assay (Peikang Biotechnology Co. Ltd.Shanghai, China)).

The skilled person will understand that whether the increased fibrindeposition and/or reduced fibrinolytic capacity is due to “local orsystemic inflammation” as used herein can be determined using one ormore biomarkers coupled to inflammation, including but not limited to Creactive protein, TNF-alpha, high sensitive C-reactive protein (hs-CRP),fibrinogen, IL-1beta, and IL-6 (e.g. by increased concentration of oneor more of these biomarkers in relation to control levels as known inthe art). Commercial analytical platforms that can be used to quantifythese biomarkers include, but are not limited to, Afinion™ (Medinor AB,Sweden), CA-7000 (Siemens Healthcare Diagnostics Inc, NY, US), Immulite®2000 Immunoassay System (Siemens Healthcare Diagnostics Inc).

Particular biomarkers that may identify local or systemic inflammationinclude high sensitive C-reactive protein (hs-CRP) (at or above 2.0 mg/lserum) and fibrinogen (at or above 3 g/l serum) (Corrado E., et al. Anupdate on the role of markers of inflammation in atherosclerosis,Journal of atherosclerosis and Thrombosis, 2010; 17:1-11, Koenig W.,Fibrin(ogen) in cardiovascular disease: an update, ThrombosisHaemostasis 2003; 89:601-9).

Unless otherwise specified, as used herein, the term “patient” includesmammalian patients (such as equines, cattle, swine, sheep, goats,primates, mice, rats, and pets in general including dogs, cats, guineapigs, ferrets, and rabbits). In particular, the term “patient” refers tohumans.

As used herein, the skilled person will understand that references toplasma will refer to the blood plasma of the patient.

As used herein, the skilled person will understand that references tothe maximum plasma concentration (or “Cmax”) of a particular substanceswill refer to the maximum concentration of that agent in blood plasma(i.e. the blood plasma of the patient). In the context of theadministration of that agent, the Cmax will refer to that occurring as adirect result of such administration (i.e. the Cmax occurring as aresult of the absorption of that agent).

As used herein, the time at which the Cmax of a particular substanceoccurs may also be referred to as the Tmax.

The skilled person will understand that the Cmax may occur at a specifictime (i.e. a particular peak in plasma concentration) or for a prolongedperiod (i.e. where the plasma concentration reaches a plateau), both ofwhich may be referred to as the time at which the Cmax occurs (theTmax). Where the Cmax occurs for a prolonged period, the time at whichthe Cmax occurs may also be taken to the mid-point of that period,although it is generally understood that the Cmax will occur as aclearly distinguishable peak at a specific time.

As described herein, the plasma concentration of PAI-1 in a patient(particularly a human) is known to follow a circadian rhythm. Typically,the maximum plasma concentration (Cmax) of PAI-1 is expected to occur ataround 06:00 hours.

Thus, references herein to the time at which the Cmax of PAI-1 occursmay be replaced with a reference to about 06:00 hours.

All absolute times (i.e. specific points in time and periods defined asbeing between specific points in time) indicated herein refer to theactual local time (i.e. the ‘clock’ time) experienced by the patient.Moreover, said times assume that the patient is adjusted to local time(for example, having had adequate time to adjust to changes in time zoneor so-called “daylight savings” time adjustments).

The skilled person will understand that the timing of the maximum plasmaconcentration of PAI-1 and compounds of the invention (or salts and/ormetabolites thereof) may be determined using techniques that are wellknown to those skilled in the art, such as by monitoring theconcentration of PAI-1 and compounds of the invention (or salts and/ormetabolites thereof) in plasma during the relevant time period.

As described herein, plasma levels of compounds of the invention (orsalts and/or metabolites thereof) may be monitored using techniqueswell-known to those skilled in the art. For example, valproate plasmalevels are determined in clinical routine e.g. by using a homogeneousenzyme immunoassay technique, based on competition of antibodies betweenvalproate in the sample and enzyme-labelled valproate added to the test(e.g. VALP2, Roche/Cobas, art nr 05108438190 (Roche DiagnosticsScandinavia AB). When the enzyme-labelled valproate is bound to theantibody, the enzyme Glucose 6-phosphate dehydrogenase, (G6PDH) isblocked and cannot consume the test enzyme substrate. Conversely, whenthe enzyme-labelled valproate is not bound to the antibody, thesubstrate is available to the enzyme and can be consumed. Theconsumption of the substrate is measured indirectly by formation of NADHfrom NAD (coenzyme reaction). NADH absorbs UV light selectively at 340nm. This means that high valproate concentration in the sample gives alarge change in absorbance at 340 nm; conversely at low valproateconcentration, there may be a small change in absorbance at 340 nm. Theconsumption of substrate gives rise to a colour change that is measuredphotochromatically at 340 and 415 nm. The absorbance is directlyproportional to the valproate concentration in the sample.

The skilled person will be able to identify compounds present in plasmaas being metabolites of compounds of the invention. Particularmetabolites of compounds of the invention that may be mentioned includethe valproate anion (e.g. metabolites that comprise a valproate anionmoiety).

The skilled person will understand that references to monitoring theplasma concentration (i.e. the blood plasma concentration in thepatient) of PAI-1 may refer to monitoring over at least one (e.g. one)24 hour period (e.g. prior to the beginning of treatment with compoundsof the invention). Such monitoring may be continuous or may involve thetaking of measurements at set intervals during this period (which maymean that, particularly in the latter case, the time between the firstand last measurement is less than 24 hours, such as around 20 hours).

The skilled person will also understand that such monitoring may insteadbe conducting for a period of time that is expected to include the Cmaxof PAI-1, as estimated by a person skilled in the art. For example,where the Cmax of PAI-1 is expected to occur at around 06:00 hours, suchmonitoring may take place at from 04:00 hours to 08:00 hours (e.g. from05:00 hours to 07:00 hours).

The timing and size of the dose of compounds of the inventionadministered will also result in low plasma concentrations of valproicacid, or a salt and/or metabolite thereof, at specific times.

Thus, in a particular embodiment of the first to seventh aspects of theinvention, administration of the compounds of the invention is such thatthe plasma concentration of valproic acid, or a salt and/or metabolitethereof, during the period from about 14:00 hours to about 18:00 hours(e.g. from about 15:00 hours to about 17:00, such as at about 16:00hours) is less than about 350 μM (such as less than about 300 μM, forexample less than about 250 μM or, more particularly, less than 200 μM,such as less than about 150 μM or less than about 100 μM).

In a more particular embodiment of the first to seventh aspects of theinvention, administration of the compounds of the invention is such thatthe plasma concentration of valproic acid, or a salt and/or metabolitethereof, during the period from about 15:00 hours to about 17:00 hours(such as at about 15:30 hours or about 16:30 hours) is less than about300 μM (such as less than about 200 μM (e.g. less than about 150 μM, orless than about 100 μM).

Further, the skilled person will be able to adjust both the timing anddose of administration of compounds of the invention in order to meetthe requirements of the timing of the Cmax and/or the presence of amaximum or minimum concentration in plasma at a specified time.

As used herein, the terms “therapeutically effective amount” and“therapeutically effective dose” refer to an amount of the active agent(i.e. the compounds of the invention) which confers the requiredpharmacological or therapeutic effect on the patient, preferably withoutundue adverse side effects. It is understood that the therapeuticallyeffective amount may vary from patient to patient.

In particular, a therapeutically effective dose of a compound accordingto the present invention is an amount sufficient to treat or prevent therelevant pathological condition and its complications, particularlywhere selected to minimise side effects (i.e. adverse events broughtabout by the action of the therapeutic agent). In view of thedisclosures herein, the skilled person will be able to adjust the doseof compounds of the invention administered in order to achieve thedesired biological effect using techniques known to those skilled in theart.

The skilled person will understand that the dose of the compounds of theinvention may be titrated such that a dose is determined that willachieve a reduction in PAI-1 plasma levels of at least about 20% (suchas at least about 30%).

In particular embodiments of the invention (for example, particularembodiments of the first to seventh aspects of the invention), the doseof the compounds of the invention is sufficient to achieve a reductionin PAI-1 plasma levels of at least about 20% (such as at least about30%), i.e. the dose is titrated to achieve the required reduction inplasma levels of PAI-1.

In more particular embodiments of the invention (for example, particularembodiments of the first to seventh aspects of the invention), the doseis sufficient to achieve a reduction in PAI-1 plasma levels of at leastabout 40% (such as at least about 50%, e.g. at least about 60%).

Similar dose titrations are known in the art and both starting dose,increments and intervals for PAI-1 measurements (generally from morningsamples), desired reduction in PAI-1 and potential dose increments maybe chosen by the person skilled in the art.

In certain embodiments, the starting doses for such dose titrations maybe in the range of e.g. 50, 100, 150, 200, 250 or 300 mg and doseincrements may be 20-100 mg every 7-28 days following a new PAI-1measurement. For example, in one such measurement the starting dose fora dose titration is 50 mg and the dose is raised in increments of 50 mgevery 7 days until a 20% reduction in circulating PAI-1 levels isachieved. In another such measurement, the starting dose for a dosetitration is 100 mg and the dose is raised in increments of 100 mg every14 days until a 20% reduction in circulating PAI-1 levels is achieved.

Without wishing to be bound by theory, it is thought that the surprisingeffects resulting from the administration of compounds of the inventionas described herein can be obtained through administration of doses thatare at a level that is not expected to result in significant levels ofadverse events.

Thus, in particular embodiments of the first to seventh aspects of theinvention, the treatment may require administering a dose of valproicacid or a pharmaceutically acceptable salt thereof (e.g. one such dosein a 24 hour period) that is selected in order to minimise the level ofadverse events resulting from such treatment (e.g. is of a sufficientlylow level to avoid the occurrence of such adverse events).

Such amounts may vary according to the frequency and mode ofadministration, the sex, age, weight and general condition of thesubject treated, the nature and severity of the condition treated and orother treatments used by the individual, and may be determined byconventional techniques in the field. The amount that is effective for aparticular therapeutic purpose will depend on the severity of thecondition as well as on the weight and general state of the subject. Itwill be understood that determination of an appropriate dosage may beachieved, using routine experimentation, by constructing a matrix ofvalues and testing different points in the matrix, all of which iswithin the ordinary skills of a person skilled in the art.

Notwithstanding the discussion of specific doses as provided herein, theskilled person will understand that the amounts of and dosage regimes ofVPA, or a pharmaceutically acceptable salt thereof, required fortreating or preventing a pathological condition associated with excessfibrin deposition and/or thrombus formation as described herein may bedetermined using the routine skill of the prescribing physician.

In particular embodiments of the first to seventh aspects of theinvention, compounds of the invention may be administered:

(i) as a single dose per 24 hour period (i.e. a single daily dose);and/or

(ii) at a total dose per 24 hour period (i.e. a total daily dose) ofabout 50 mg to about 1000 mg (particularly about 200 mg to about 600 mg,such as about 300 mg to about 500 mg).

More particularly, the single daily dose described above (e.g. at point(i) directly above) may be administered at a time from about 20:00 hoursto about 06:00 hours.

In a more particular embodiment, the single daily dose (e.g. describedat point (i) above) may be administered at a time from about 21:00 hoursto about 05:00 hours (e.g. about 22:00 hours to about 04:00 hours).

In a yet more particular embodiments (particularly wherein the treatmentis administered as a pharmaceutical composition that is not formulatedfor delayed release of the active ingredient), the single daily dose(e.g. described at point (i) above) may be administered at a time fromabout 02:00 hours to about 06:00 hours (e.g. about 03:00 hours to about05:00 hours, such as about 04:00 hours).

In further particular embodiments (particularly wherein the treatment isadministered as a pharmaceutical composition that is formulated fordelayed release of the active ingredient, such as those described in theeight aspect of the invention herein), the single daily dose (e.g. asdescribed at point (i) above) may be administered at a time from about20:00 hours to about 00:00 hours (e.g. about 21:00 hours to about 23:00hours, such as at about 22:00 hours).

In alternative embodiments (particularly wherein the treatment isadministered as a pharmaceutical composition that is formulated fordelayed release of the active ingredient, such as those described in theeight aspect of the invention herein), the single daily dose (e.g. asdescribed at point (i) above) may be administered prior to sleep (i.e.immediately before the patient begins to attempt to sleep, which mayalternatively be described as “before bed”, “before sleep”, or thelike).

In particular embodiments of the invention (for example, particularembodiments of the first to seventh aspects of the invention), compoundsof the invention may be administered in a manner such that the plasmaconcentration of valproic acid, or a salt and/or metabolite thereof,during a particular period (e.g. a 24 hour period) mimics the plasmaconcentration of PAI-1 during the same period.

As used herein, references to a plasma level that “mimics” another willbe understood to mean that the relative plasma levels of the two agentsfollow substantially similar patterns of variation (e.g. the curvesobtained by plotting the plasma concentrations of the two agents may besubstantially superimposable, although the absolutelevels/concentrations of the two agents may be different). The term“mimics” has its ordinary meaning in the art, i.e. to resemble,simulate, approximate, follow or impersonate, but not necessarilyreplicate exactly or precisely.

The skilled person will understand that, in addition to the eveningdose, a lower morning dose may be administered, which dose would beabsorbed when the PAI-1 level starts to increase in the late afternoon.For example, in one such treatment 10-500 mg (such as 50-300 mg, moreparticularly 100 or 200 mg) is administered approximately 10-14 hours(such as e.g. 12 hours) after the evening dose.

Thus, in a particular embodiment of the invention, a lower morning doseis administered, in addition to the evening dose, which dose willconsist of about 10 to about 500 mg (such as about 50 to about 300 mg,more particularly about 100 or about 200 mg) that is administered duringa time period that is about 10 to about 14 hours (such as e.g. about 12hours) after the evening dose. In a specific embodiment, this morningdose is about 20 to about 50% (such as about 20, about 30 or about 40%)of the evening dose.

In a more particular embodiment, there is provided a once-dailyformulation that provides the same effect as the morning and eveningdose described in the embodiment directly above, which may be providede.g. in the form of a dual layer formulation with a core giving a secondsmall peak coinciding with the rise in PAI-1, or with differently coatedand/or formulated granules formulated for such a release profile.

As described herein, it has been found that valproic acid (VPA) potentlyreduces plasma PAI-1 levels, with such reduction allowing for anincrease in the activity of endogenous t-PA. In particular,administration of VPA such that plasma levels thereof coincide with peakplasma levels of PAI-1 allows for the treatment or prevention ofpathological conditions associated with excess fibrin deposition and/orthrombus formation.

Thus, references herein (e.g. in the first to seventh aspects of theinvention) to uses in treating or preventing a pathological conditionassociated with excess fibrin deposition and/or thrombus formation mayalso refer to treating or preventing a pathological condition expectedto benefit from (i.e. be treated or prevented by) reduced activity ofPAI-1.

For the avoidance of doubt, specific conditions referred to as beingassociated with excess fibrin deposition and/or thrombus formation, asknown to the skilled person (in particular, as described herein), mayalso be understood to be expected to benefit from (i.e. be treated orprevented by) reduced PAI-1 activity, which may be understood to resultfrom reduced levels of PAI-1 in plasma.

In particular, in a further aspect of the invention, there is provided amethod of reducing PAI-1 levels (i.e. levels of PAI-1 in plasma) in apatient in need thereof comprising the step of administering atherapeutically effective amount of valproic acid, or a pharmaceuticallyacceptable salt thereof.

Similarly, specific methods of treating or preventing conditionsassociated with excess fibrin deposition and/or thrombus formation asreferred to herein may also be understood as being methods of reducingPAI-1 levels in a patient in need thereof.

For example, in a yet further alternative first aspect of the invention,there is provided a method of reducing PAI-1 levels in a patient in needthereof comprising administering at least one dose of a therapeuticallyeffective amount of valproic acid, or a pharmaceutically acceptable saltthereof, to a patient such that the maximum plasma concentration (Cmax)of valproic acid, or a salt and/or metabolite thereof, in the patientoccurs during a time period that is from four hours before to one hourafter the maximum plasma concentration (Cmax) of PAI-1 in the patient.

As used herein, references to reducing levels of PAI-1 (and, similarly,to reduced (or inhibited) PAI-1 activity, e.g. references to inhibitingPAI-1) may refer to levels of PAI-1 in plasma during treatment withcompounds of the invention being at (e.g. reduced to or maintained at)levels lower than (e.g. at least 10% lower than, such as at least 20%lower than, for example at least 30%, at least 40%, at least 50% or atleast 60%) levels of PAI-1 occurring prior to treatment with compoundsof the invention.

Compounds of the Invention

Again, as indicated herein, the term “compounds of the invention” refersto valproic acid and pharmaceutically acceptable salts thereof,including mixtures thereof. The skilled person will understand thatvalproic acid may also be referred to as, inter alia, 2-propylpentanoicacid and VPA.

The compounds presented herein include, where relevant, alldiastereomeric, enantiomeric, and epimeric forms. For compoundsdescribed herein that exist as tautomers, all tautomers are includedwithin the formulas described herein. Further, the compounds describedherein may be formed as, and/or used as, salts (e.g. pharmaceuticallyacceptable salts). The skilled person will understand that referencesherein to salts of compounds will include references to pharmaceuticallyacceptable salts.

Compounds described herein may be prepared using techniques andprocedures known to those skilled in the art. Exemplary syntheticmethods useful for synthesizing the compounds in the applicationinclude, for example, those disclosed in Nogrady (1985) MedicinalChemistry A Biochemical Approach, Oxford University Press, New York,pages 388-392; Silverman (1992); Fieser and Fieser's Reagents forOrganic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd'sChemistry of Carbon Compounds, Volumes 1-5 and Supplementals (ElsevierScience Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wileyand Sons, 1991), March's Advanced Organic Chemistry, (John Wiley andSons, 4th Edition) and Larock's Comprehensive Organic Transformations(VCH Publishers Inc., 1989).

VPA may be commercially available, for example from Sigma-Aldrich (underproduct number P4543 as at 1 Oct. 2014). Pharmaceutically acceptablesalts of VPA (such as sodium salt thereof) may also be commerciallyavailable. It will also be appreciated that VPA, or pharmaceuticallyacceptable salts thereof, may be synthesised using, techniques wellknown to those skilled in the art.

As described herein, VPA may be formulated and/or administered in theform of a pharmaceutically acceptable salt thereof.

The skilled person will understand that pharmaceutically acceptablesalts (e.g. of VPA) may include but are not limited to:

(a) salts formed when an acidic proton is replaced by a metal ion, suchas for example, an alkali metal ion (e.g. lithium, sodium, potassium),an alkaline earth ion (e.g. magnesium, or calcium), or an aluminium ion,or is replaced by an ammonium cation (NH₄ ⁺);(b) salts formed by reacting VPA with a pharmaceutically acceptableorganic base, which includes alkylamines, such as ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine,dicyclohexylamine, tris(hydroxymethyl)methylamine, and salts with aminoacids such as arginine, lysine, and the like;(c) salts formed by reacting VPA with a pharmaceutically acceptableacid, which provides acid addition salts. Pharmaceutically acceptableacids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitricacid, phosphoric acid, metaphosphoric acid, and the like; or with anorganic acid, such as, for example, acetic acid, propionic acid,hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid,lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, trifluoroacetic acid, tartaric acid, citric acid, benzoicacid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid, toluenesulfonicacid, 2-naphthalenesulfonic acid,4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid,4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuricacid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylicacid, stearic acid, muconic acid, and the like.

Particular pharmaceutically acceptable salts of VPA that may bementioned include those mentioned at point (a) above. More particularpharmaceutically acceptable salts of VPA that may be mentioned includethose where the carboxylic acid proton is replaced with an alkalineearth ion (e.g. magnesium or calcium) or, more particularly, an alkalimetal ion (e.g. lithium, sodium or potassium).

In particular embodiments of each aspect of the invention, VPA isadministered and/or formulated (as appropriate) in the form of thesodium salt thereof (i.e. sodium valproate). In more particularembodiments, VPA is administered and/or formulated (as appropriate) inthe form of a mixture of VPA (i.e. in the non-salt form) and the sodiumsalt thereof (i.e. sodium valproate), such as an equal mixture thereof.

For instance in particular embodiments of the invention (i.e. eachaspect of the invention), VPA may administered and/or formulated (asappropriate) in the form of a mixture of the sodium salt thereof (i.e.sodium valproate) and valproic acid. Several such mixtures are known inthe art, such as: valproate semisodium, also known as divalproex sodium(1:1 molar relationship between valproic acid and sodium valproate),which is marketed, for example, as Depakote and Depakote ER (by AbbVieInc.); and valproate sodium (1:2.3 ratio between valproic acid andsodium valproate), which is marketed, for example, as Epilex Chrono.

Additional pharmaceutically acceptable salts that may be mentionedinclude those described in Berge et al., J. Pharm. Sci. 1977, 66, 1-19;and “Handbook of Pharmaceutical Salts, Properties, and Use”, Stah andWermuth, Ed.; Wiley-VCH and VHCA, Zurich, 2002 (the contents of whichare incorporated herein in their entirety).

References to “salts” of compounds of the invention will be understoodto refer to salt forms that may occur through exchange of anions orcations with compounds of the invention, for example, in blood plasma.In particular, the term “salts” may also refer to pharmaceuticallyacceptable salts, such as those described herein.

As described herein, VPA may also be formulated and/or administered inthe form of a prodrug thereof, or a pharmaceutically acceptable salt ofsaid prodrug.

As used herein, the term prodrug when used in relation to VPA will beunderstood to refer a compound that may be converted to VPA in vivo(i.e. following administration).

Such prodrugs may be identified by a person skilled in the art and mayinclude ester (e.g. methyl or ethyl ester) or amide derivatives of VPA.Particular prodrugs that may be mentioned include 2-propylpentanamide(also known as valpromide).

When compounds of the invention are administered in the form of aprodrug thereof, the skilled person will be able to adjust the doseadministered in order to achieve the equivalent dose of VPA as required.

Commercially-available products containing valproic acid and/or sodiumvalproate, or prodrugs thereof, include but are not limited to:

Depakote (AbbVie Inc.), Absenor (Orion Corporation), Convulex (Pfizer),Convulex CR, Depakene/Depakine/Depalept/Deprakine (AbbVie Inc./SanofiAventis), Depakine Chrono (Sanofi), Depakene-R (Kyowa Hakko Kogyo),Selenica-R (Kowa), Encorate (Sun Pharmaceuticals India), Encorate Chrono(Sun Pharmaceuticals), Epival (Abbott Laboratories), Epilim (Sanofi),Epilim Chronospheres modified release granules, Epilim Chrono Controlledrelease tablets, Epilim Chrono Prolonged release tablets, Stavzor (NovenPharmaceuticals), Valcote (Abbott Laboratories), Valpakine (SanofiAventis), Depamide (Sanofi-Avetis), Dipexil-R (Bial), Eliaxim (Bial),Sodium Valproate Sandoz Tablets (Sanofi), Vaipro Tablets (Alphapharm),Valproate Winthrop Tablets (Sanofi), Valprease (Sigma), Epilim ECmodified release tablets (Sanofi-Aventis), Oriept (Wockhardt), EpilimChrono (Sanofi) (1:2.3 ratio of valproic acid and sodium valproate),Epilim EC200 (Sanofi), Valprol CR (Intas Pharmaceutical), Episentaprolonged release (Beacon), Valproic Acid capsules, USP (Teva), Stavzor(Noven), Orfiril (Desitin Pharmaceuticals).

Commercially-available products containing valproic acid and/or sodiumvalproate, or prodrugs thereof, will also include generic version of theabove-mentioned formulations, which may be sold/marketed under adifferent name.

Administration of the Compounds

The skilled person will understand that there is also provided apharmaceutical composition comprising valproic acid, or apharmaceutically acceptable salt thereof, and optionally comprising oneor more pharmaceutically acceptable excipient, for use in treating orpreventing a pathological condition associated with excess fibrindeposition and/or thrombus formation as described in first to seventhaspects of the invention (including all embodiments thereof).

Compounds of the invention may be administered to a subject in aconvenient manner such as by the oral, intravenous, intramuscular,subcutaneous, intraperitoneal, intranasal, buccal, transdermal,intradermal, or suppository routes as is known in the art. Inparticular, compounds of the invention may be administered by the oralroute; for example, as a pharmaceutical formulation suitable for oraladministration (e.g. a tablet, capsule, buccal film, spray or the like).

In particular, pharmaceutical formulations suitable for oraladministration may be presented as discrete units, such as capsules ortablets (e.g. tablets), which each contain a predetermined amount of theactive ingredient, and which may include a suitable excipient.Furthermore, the orally available formulations may be in the form of apowder or granules, a solution or suspension in an aqueous ornon-aqueous liquid, or an oil-in-water or water-in-oil liquid emulsion.

Compositions intended for oral use may be prepared according to anyknown method, and such compositions may contain one or more agentsselected from the group consisting of sweetening agents, flavouringagents, colouring agents and preserving agents in order to providepharmaceutically elegant and palatable preparations.

For example, tablets may contain the active ingredient(s) in admixturewith non-toxic pharmaceutically acceptable excipients which are suitablefor the manufacture of tablets. These excipients may, for example, be:inert diluents, such as calcium carbonate, sodium carbonate, lactose,calcium phosphate or sodium phosphate; granulating and disintegratingagents, for example corn starch or alginic acid; binding agents, forexample, starch, gelatine or acacia; and lubricating agents, for examplemagnesium stearate, stearic acid or talc. The tablets may be uncoated orthey may be coated by known techniques to delay disintegration andabsorption in the gastrointestinal tract and thereby provide a sustainedaction over a longer period. For example, a time delay material such asglyceryl monostearate or glyceryl distearate may be employed. They mayalso be coated by the techniques described in U.S. Pat. Nos. 4,356,108;4,166,452; and 4,265,874, the contents of which are incorporated hereinby reference, to form osmotic therapeutic tablets for controlledrelease.

Moreover, formulations for oral use may also be presented as hardgelatine capsules where the active ingredient is mixed with an inertsolid diluent, for example, calcium carbonate, lactose, calciumphosphate or kaolin, or a soft gelatine capsules wherein the activeingredient is mixed with water medium (such as a water miscible liquide.g. poly ethylene glycol) or an oil medium, for example peanut oil,liquid paraffin, or olive oil. Such gelatine capsules may be formulatedto contain granules of the active ingredient, which granules may beformulated (e.g. coated) in a manner as described herein for tablets.

Further, formulations for oral use may be presented into the form oftablets composed of compressed microparticles (e.g. granules), whichmicroparticles may be individually coated.

Thus, in embodiments wherein the formulation comprises microparticulates(e.g. in a capsule or tablet, such as a tablet composed of compressedmicroparticles or capsules containing granules), such microparticles mayhave different coatings (or formulated for delayed release usingpolymers as described below), which coatings/formulations may beselected to regulate the release of compounds of the invention; forexample, in order to control absorption and render a plasma profilemimicking the PAI-1 plasma profile. The use of suchcoatings/formulations to control absorption/release of a drug is knownin the art and can e.g. be based on different polymers e.g. based onacrylic acid or cellulose and is described more extensively below.

Multiple unit dosage forms are less dependent on the degree of fillingof the stomach and may therefore lead to lower variability in e.g.absorption profiles in different patients.

The single compartments of multiple unit dosage forms can be prepared bycommonly known methods including granulation, pelletizing, extrusion,hot melt extrusion, tableting and/or coating techniques. For examples onthe production of tablets and/or capsules from coatedgranules/microtablets see e.g. WO 96/01621, WO 96/01624, Siddique,Khanam and Bigoniya, AAPS PharmSciTech 2010. These references alsoprovide information on how different materials can be used to controlthe release of drug from a tablet or capsule (or from granules in saidtablet or capsule).

In particular, the skilled person will be aware that valproic acid is aliquid and sodium valproate is hygroscopic. Suitable excipients andpreparation processes for these types of ingredients are known in theart and include e.g. coating of components with a suitable polymer (e.g.methacrylic acid copolymers of different types) and/or water insolublematerials such as waxes/fatty acids etc., in order to achieve reducedhygroscopicity. Such polymers may also be used to delay the releaseand/or absorption of the drug according to the invention.

For buccal and sublingual use, creams, ointments, jellies, solutions ofsuspensions and the like containing the compounds of the invention maybe employed.

Pharmaceutical compositions may also be in the form of suppositories forrectal administration of the compounds of the invention. Thesecompositions can be prepared by mixing the compounds of the inventionwith a suitable non-irritating excipient which is solid at ordinarytemperatures but liquid at the rectal temperature and will thus melt inthe rectum to release the drug. Such materials include, for example,cocoa butter and polyethylene glycols.

Pharmaceutical compositions comprising compounds of the invention mayalso be provided in the form of liposome delivery systems, such as smallunilamellar vesicles, large unilamellar vesicles, and multilamellarvesicles. Liposomes may be formed from a variety of phospholipids, suchas cholesterol, stearylamine, or phosphatidylcholines.

Pharmaceutical forms suitable for injectable use include, but is notlimited to, sterile aqueous solutions (where water soluble) ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. In all cases the form mustbe sterile and must be fluid to the extent that easy syringabilityexists. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, sterile water, ethanol,polyol (for example, glycerol, propylene glycol and liquid polyethyleneglycol and the like), suitable mixtures thereof, and vegetable oils. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of a dispersion, and by the use of surfactants. The preventionsof the action of microorganisms can be brought about by variousantibacterial and antifungal agents; for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate, andgelatin.

Sterile injectable solutions are prepared by incorporating the activematerial in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredient into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze-dryingtechnique, which yield a powder of the active ingredient plus anyadditional desired ingredient from previously sterile-filtered solutionthereof.

We have found that compounds of the invention may be convenientlyadministered to a subject by the oral route, particularly in the form ofa tablet or capsule (e.g. a tablet). Moreover, we have found that theparticular dosage regimes contemplated in the invention are particularlysuited to oral administration in the form of a tablet or capsule that isformulated such the release of compounds of the invention from saidtablet or capsule after oral administration is delayed.

As used herein, references to formulations allowing for delayed orcontrolled released will be understood by those skilled in the art. Inthis regard, it will be understood that the terms delayed and controlledmay be used interchangeably.

In an eighth aspect of the invention, there is provided a pharmaceuticalcomposition comprising valproic acid, or a pharmaceutically acceptablesalt thereof, wherein the composition is in the form of a tablet orcapsule for oral administration and is formulated such thatsubstantially all of the valproic acid, or a pharmaceutically acceptablesalt thereof, is released during a period from about four to about eighthours after administration.

As used herein, references to a capsule will include capsules filledwith the active ingredient in powder form, or in the form of granulesand/or microparticles, which granules and/or microparticles may becoated as described herein, and which capsule may itself be coated.Furthermore, the granules may be formulated for specific releaseprofiles using e.g. different delayed/controlled release polymers(and/or coating the granules).

As used herein, references to a tablet will include tablets formed fromcompressed granules and/or microparticles, which granules and/ormicroparticles may be coated as described herein, and which tablet mayitself be coated.

As used herein (particularly in reference to the eight aspect of theinvention, including all embodiments thereof), the term “substantiallyall” will refer to an amount that is at least 60% of the total amountpresent (i.e. the total amount included in the composition). Inparticular, the term may refer to an amount that is at least 70% of thetotal, such as at least 80% of the total. More particularly, the termmay refer to an amount that is at least 90% of the total, such as atleast 95% (e.g. at least 99%) of the total.

In a particular embodiment of the eighth aspect of the invention,references to substantially all of the valproic acid, or apharmaceutically acceptable salt thereof, being released may refer tosubstantially all of one dose (i.e. at least one therapeuticallyeffective dose) thereof.

The skilled person will understand that the release of the activeingredient may be delayed if the composition is administered with orshortly after food. Thus, references to the time taken for the activeingredient to be released may refer to the time taken for such releasewhen the composition is administered to a patient at least two hoursafter that patient has consumed food (which may be referred to asadministration on an empty stomach, or the like).

It may also be appreciated that it may be beneficial to administercompounds of the invention with food (e.g. to reduce gastrointestinalside-effects). Thus, in a particular embodiment of the first to seventhaspects of the invention, the treatment comprises administering thevalproic acid, or a pharmaceutically acceptable salt thereof, with food(e.g. administered to a patient who has consumed food less than twohours prior to administration or who will be directed to consume foodwithin 30 minutes of administration).

As used herein (particularly in reference to the eight aspect of theinvention, including all embodiments thereof), references to an activeingredient being “released” (i.e. from a pharmaceutical formulation)will refer to the active ingredient being in a form that is availablefor absorption (i.e. when administered orally, systemic absorption fromthe gastro intestinal (GI) tract). When used in relation to tabletsand/or capsules for oral administration, the term will indicate that theactive ingredient is not contained in said tablet or capsule (which mayinclude the active ingredient being no longer contained within granules(e.g. coated granules) and/or microparticles contained within saidtablets or capsules) but is instead distributed in the GI tract.

In a particular embodiment of the eighth aspect of the invention, thepharmaceutical composition is formulated such that substantially all ofthe valproic acid, or a pharmaceutically acceptable salt thereof, isreleased during a period from about six to about eight hours afteradministration (such as about six to about seven hours afteradministration, or such as about seven to about eight hours afteradministration, e.g. about seven hours after administration).

In more particular (and alternative) embodiments of the eighth aspect ofthe invention, the pharmaceutical composition is formulated such thatsubstantially all of the valproic acid, or a pharmaceutically acceptablesalt thereof, is released during a period that is:

(i) from about three to about five hours after administration (fromabout four to about five hours after administration);

(ii) from about four to about six hours after administration;

(iii) from about five to about seven hours after administration;

(iv) from about six to about eight hours after administration; or

(v) from about eight to about ten hours after administration.

In a particular embodiment of the eighth aspect of the invention, thepharmaceutical composition may further comprise one or morepharmaceutically acceptable excipient (e.g. a pharmaceuticallyacceptable adjuvant, diluent or carrier), such as those describedherein. In such embodiments, the compounds of the invention may beprovided in admixture with said one or more pharmaceutically acceptableexcipient.

The skilled person will understand that pharmaceutical formulations(i.e. tablets or capsules) comprising compounds of the invention (suchas those described in the eight aspect of the invention, includingembodiments thereof) will contain all or part of a therapeuticallyeffective dose of the compounds of the invention.

For the avoidance of doubt, such a dose may be provided in a single unitof the composition (e.g. a single tablet or capsule), or may be providedby the combined administration of several units of the formulation eachcomprising a corresponding fraction of the dose (e.g. two tablets eachcontaining half of the required dose, or a plurality of microparticleseach containing the requisite fraction of the required dose).

In particular, said formulations (e.g. tablets for oral administration)may comprise a single therapeutically effective dose. Thus, inparticular embodiments of the eight aspect of the invention, thecomposition comprises a dose (e.g. a total daily dose) of valproic acid,or a pharmaceutically acceptable salt thereof, as defined in any one ormore of the first to seventh aspects of the invention (including allembodiments thereof).

Depending on the dose required, pharmaceutical formulations that may bementioned include those in which the active ingredient is present in atleast 1% (or at least 10%, at least 30% or at least 50%) by weight. Thatis, the ratio of active ingredient to the other components (e.g. thepharmaceutically acceptable excipient) of the pharmaceutical compositionis at least 1:99 (or at least 10:90, at least 30:70 or at least 50:50)by weight.

Thus, the skilled person will understand that the invention furtherprovides a process for the preparation of pharmaceutical formulations asdescribed herein (such as those described in the eight aspect of theinvention, including embodiments thereof), which process comprisesformulating compounds of the invention in a manner as described herein.In particular, such a process may comprise the steps of:

(a) bringing compounds of the invention into association with one ormore pharmaceutically acceptable excipient (e.g. to form an admixturethereof); and

(b) formulating as a tablet or capsule (as described herein).

The skilled person will understand that the term bringing intoassociation means that the relevant components are rendered suitable foradministration in conjunction with each other.

As described herein, compounds of the invention may be administeredand/or formulated in a form coated by, or administered with, a materialto delay release of the active ingredient. In particular, formulationsin the form of a tablet may be coated with such a material and/orformulated with polymers that regulate the release. Moreover,formulation in the form of a capsule may be formulated such the capsuleis composed of, or comprises an amount (i.e. an effective amount) of,such a material.

Thus, pharmaceutical compositions of the eight aspect of the inventionmay be referred to as “delayed release” or “controlled release”compositions or formulations, or the like.

In such instances, the skilled person will understand that the materialto delay release of the active ingredient will be selected and/orformulated in a manner to delay release of the active ingredient for therequired time (e.g. for about six hours).

The skilled person will be familiar with materials used to delay (i.e.for delaying) the release of active ingredients, particularly whenadministered in the form or oral compositions (such as tablets andcapsules). Such materials may be described in, for example, Remington'sPharmaceutical Science and U.S. Pharmacopeia (The United StatesPharmacopeia-National Formulary (USP-NF)), Remington: The Science andPractice of Pharmacy, 19th Ed. (Easton, Pa.: Mack Publishing Company,1995); Hoover, John E., Remington's Pharmaceutical Sciences, MackPublishing Co., Easton, Pa. 1975; Liberman, H. A. and Lachman, L., Eds.,Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; andPharmaceutical Dosage Forms and Drug Delivery Systems, 7th Ed.(Lippincott Williams Wilkins 1999), the contents of which areincorporated herein in their entirety.

For example, materials used to delay the release of active ingredientsmay include sustained release polymers, such as hydroxypropyl cellulose,hydroxypropyl methylcellulose, carboxymethyl cellulose, chitosan, aloemucilage, pectin, ethyl cellulose, polyvinyl chloride, polyethylene andpolyvinylpyrrolidone (PVP) (e.g. hydroxypropyl cellulose, hydroxypropylmethylcellulose, carboxymethyl cellulose, chitosan, aloe mucilage,pectin, ethyl cellulose, polyvinyl chloride and polyethylene). Moreover,one way of achieving a sustained release coating is to mix a watersoluble polymer such as HPMC with a water insoluble polymer such asethyl cellulose. The skilled person will understand that differentmaterials used and different ratios thereof will result in differentrelease patterns, and will be able to adjust the formulation accordingly(i.e. to achieve the desired release profile).

The skilled person will understand that where compositions areadministered and/or formulated in a form coated by, or administeredwith, a material to delay release of the active ingredient, saidmaterial may be composed of more than one pharmaceutically acceptablesubstance (e.g. one or more pharmaceutically acceptable coating). Forexample, where compositions of the eight aspect of the invention areadministered in the form of a tablet, said tablet may comprise one ormore pharmaceutically acceptable coatings of a material to delay releaseof the active ingredient.

In such instances, the skilled person will understand that the delay ofthe release of the active ingredient from the composition (e.g. thetablet) is achieved as a combined effect of these coatings. For example,where a tablet is coated so as to delay release for a total of six hoursafter oral administration, the tablet may comprise two layers ofcoating, each coating delaying release for three hours (or one coatingdelaying release for two hours and a further coating delaying releasefor four hours), i.e. with the first coating being removed to expose thesecond coating, and so on (in other words, said coatings being exposedin a sequential manner).

In particular embodiments of the eight aspect of invention, wherecompositions of the eight aspect of invention comprise one or morecoatings (e.g. are in the form of a coated tablet), one or more of saidcoatings may be a coating for preventing release of the activeingredient, or preventing exposure of further coatings, in the stomach.In particular, one or more (e.g. one) of said coatings may be an entericcoating. Said enteric coatings will be well known to the person skilledin the art.

In certain embodiments of the eight aspect of invention (particularlythose referring to tablets having one or more coating), the corecomponent (e.g. the core component of a coated tablet) may contain oneor more components designed to promote disintegration in aqueous media.

Thus, in a particular embodiment of the eight aspect of the invention,the formulation is provided as a tablet (or capsule) for oraladministration comprising one or more coated core (e.g. a single coatedcore, or a plurality of coated granules or microparticles each havingsuch a core), said core(s) containing valproic acid or apharmaceutically acceptable salt thereof, wherein:

(i) said coating is formed of material selected and/or formulated in amanner to delay release of the active ingredient for the required time(e.g. for about six hours); and

(ii) said core is formulated to in a manner designed promotedisintegration in aqueous media (e.g. comprising one or moredisintegrants).

Such disintegrants will be well known to those skilled in the art,including agents designed to swell upon contact with aqueous media.

The skilled person will understand that there are several materials thatcan be used to form an enteric coating on a tablet/capsule and/orgranules/microtablets/pellets/multiparticulate/multiple unit dosageforms. These include but are not limited to shellac, waxes, fatty acids,polymers, plastics and plant fibers.

Examples of such polymers include, but are not limited to, hypromellosephthalate (hydroxypropyl methylcellulose phthalate, HPMCP), hypromelloseacetate succinate, cellulose acetate trimellitate, acrylicacid/methacrylic acid copolymers (e.g. poly(methacrylic acid-co-methylmethacrylate), cellulose acetate phthalate (CAT), poly(vinyl acetatephthalate, PVAP) and ethyl acrylate. Other materials for enteric coatinginclude dextrins, amylose starch and starch derivatives, sodiumalginate, Zein and Aqua-Zein R.

Commercially available systems for enteric coatings and coatings forsustained release include variants of OPADRY® (Colorcon), Titancoat,Kollicoat® (BASF), Eudragit®, (e.g. Eudragit® RL, Eudragit® RS,Eudragit® S, Eudragit® L and Eudragit® E), Sheffcoat EC and SheffcoatEnt.

The skilled person will understand that different materials havedifferent properties e.g. when it comes to the dissolution pH and canthus be used to control the absorption pattern, e.g. delaying release ofa drug for a specific time, by a person skilled in the art. In addition,the thickness of the coating can also be altered to achieve a specificpattern. Furthermore, if coated granules are used e.g. in a capsule orcompressed tablet, different coatings (and/or coating thicknesses) canbe used in order to mimic the pattern of PAI-1 plasma concentration forthe compounds of the invention. More specifically, a combination ofseveral (e.g. 2-5) differently coated granules/microparticles may beused to achieve the desired effect in mimicking the pattern of PAI-1plasma concentration for the compounds of the invention.

One way of extending the delay in absorption of an enteric coating is tomix an enteric coating polymer with a smaller amount of a sustainedrelease polymer; as described in e.g Tirpude and Puranik, J Adv PharmTechnol Res 2011, where 10% of sustained release acrylic polymers(Eudragit NE30D) was mixed with 90% enteric acrylic polymers (EudragitL30D555). Thus, materials such as polymers with different dissolutioncharacteristics may be combined in different ratios to achieve a desiredpattern of absorption according to the invention. Other examples ofmethods to achieve different absorption patterns by using various gradesof hydrophilic polymers and how to make matrix tablets from granules aredescribed in Roy, Brahma, Nandi and Panda, Int J Appl Basic Med Res.2013.

Different ways to achieve controlled release using matrix tablets anddescription of different polymers and matrices is also described inhttp://www.pharmainfo.net/reviews/matrix-tablets-important-tool-oral-controlled-release-dosage-forms,the disclosures of which are incorporated herein by reference in theirentirety.

For more details on enteric coatings see e.g. Singh Deep Hussan et at,IOSR Journal of Pharmacy (2012), and the Handbook of PharmaceuticalExcipients Rowe, Raymond C; Sheskey, Paul J; Cook, Walter G; Fenton,Marian E., Seventh edition, the disclosures of which are incorporatedherein by reference in their entirety.

Some coatings may require the use of plasticizer/s to obtain goodresults and the use of such agents is known in the art. Suchplasticizers include e.g. citrate esters, glycerol, propylene glycol,diethyl phthalate, dibutyl phthalate, dibutyl sebacate, tributulcitrate, acetylated monoglycerides, triacetin and glycerintriacetate.

Pigments and/or these plasticizers may be added to e.g. a polymericsolution in order to improve the technical properties of e.g. a membraneor modify the release characteristics.

Compounds of the invention may be coated by, or administered with, amaterial to prevent their inactivation. For example, the active materialmay be administered in an adjuvant, co-administered with e.g. enzymeinhibitors or in liposomes. Adjuvants contemplated herein include, butare not limited to, resorcinols, non-ionic surfactants such aspolyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzymeinhibitors include; but are not limited to, pancreatic trypsininhibitor, diisopropylfluorophosphate (DFP) and trasylol. Liposymesinclude water-in-oil-in-water P40 emulsions as well as conventionalliposomes Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms.

As described herein, the skilled person will understand that whenadministered orally the active compound may be combined with an inertdiluent or with an edible carrier, or it may be enclosed in hard or softshell gelatin capsule, or it may be compressed into tablets, or it maybe incorporated directly with the food of the diet. For oral therapeuticadministration, the active material may be incorporated with excipientsand used in the form of ingestible tablets, buccal tablets, troches,capsules, elixirs, suspensions, syrups, wafers, and the like. Inaddition, the active material may be incorporated into sustained-releasepreparations and formulations. For example, the active material may beincorporated in enterotablets/capsules and/or bi-phasic releaseformulations, which formulations will be known to the skilled person.For example, bi-phasic release formulation may be of the type describedin US2007/0232528A1 (the contents of which are incorporated herein intheir entirety), which formulations may be suitable for administrationduring a period from about 22:00 to 00:00 hours (e.g. about 23:00hours).

As used herein, the term “pharmaceutically acceptable excipient” willinclude pharmaceutically acceptable adjuvants, diluents and carriers, asknown to those skilled in the art. This may include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like.

Thus, the tablets, troches, pills, capsules, and the like may alsocontain the following: a binder such as gum tragacanth, acacia, cornstarch, or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such as sucrose, lactose or saccharin may be added or a flavoringagent such as peppermint, oil of wintergreen, or cherry flavoring. Whenthe dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both. A syrup or elixir may contain the activecompound, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye, and flavoring such as cherry or orange flavor. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. The use of such excipients is well known in the art; see, forexample, Remington's Pharmaceutical Science and U.S. Pharmacopeia (TheUnited States Pharmacopeia-National Formulary (USP-NF)), Remington: TheScience and Practice of Pharmacy, 19th Ed. (Easton, Pa.: Mack PublishingCompany, 1995); Hoover, John E., Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. and Lachman, L.,Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980;and Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Ed.(Lippincott Williams Wilkins 1999).

As described herein, tablets and/or capsules formulated to delay therelease of compounds of the invention from said tablet after oraladministration (as described in the eight aspect of the invention, andembodiments thereof) are particularly suited for use in treating orpreventing a pathological condition associated with excess fibrindeposition and/or thrombus formation in accordance with the particulardosage regimes described herein.

Thus, in a ninth aspect of the invention, there is provided apharmaceutical composition as described in the eight aspect of theinvention (including any one or more embodiments thereof) for use intreating or preventing a pathological condition associated with excessfibrin deposition and/or thrombus formation, wherein said treatment isas described in any one of the first to seventh aspects of the invention(including any one or more embodiments thereof).

In an alternative ninth aspect of the invention, there is provided theuse of a pharmaceutical composition as described in the eight aspect ofthe invention (including any one or more embodiments thereof) in themanufacture of a medicament for use in treating or preventing apathological condition associated with excess fibrin deposition and/orthrombus formation, wherein said treatment is as described in any one ofthe first to seventh aspects of the invention (including any one or moreembodiments thereof).

In a further alternative ninth aspect of the invention, there isprovided a method of treating or preventing a pathological conditionassociated with excess fibrin deposition and/or thrombus formation in apatient in need thereof as described in any one of the first to seventhaspects of the invention (including any one or more embodimentsthereof), wherein the valproic acid or pharmaceutically acceptable saltthereof is administered in the form of a pharmaceutical composition asdescribed in the eight aspect of the invention (including any one ormore embodiments thereof).

As described herein, the skilled person will be able to adjust theformulation and manner of administration of compounds of the inventionin order to achieve the desired parameters, such as the desired timingand/or levels of plasma concentrations of specific agents.

For instance, the skilled person will be aware that various formulationsof compounds of the invention are commercially available and may beadministered in a manner suitable for use in, inter alia, treatments asdescribed in the first to seventh aspects of the invention.

Thus, in particular embodiments of invention (for example, particularembodiments of the first to seventh and ninth aspects of the invention)there is provided valproic acid, or a pharmaceutically acceptable saltthereof, for use in treating or preventing a pathological conditionassociated with excess fibrin deposition and/or thrombus formation,wherein said treatment comprises administering a pharmaceuticalcomposition comprising a dose of valproic acid, or a pharmaceuticallyacceptable salt thereof, to a patient in a form (i.e. a specificformulation), and at a specific dose and time, as indicated in thefollowing table.

Formulation name Dose of active agent Time of administration Depakote125 to 750 mg (e.g. 250 Approximately 22:00 hours mg or 500 mg) once to00:00 hours (e.g. about daily 23:00), or before sleep. If administeredwith food absorption may be delayed and the drug can be administeredfrom approximately 19:00 to 21:00 hours. Depakote ER 250 to 750 mg (e.g.250 Approximately 18:00 to 5or 00 mg) once daily 21:00 (e.g. about19:00), or before sleep. Depakote 125 to 750 mg (e.g. 250 Approximately22:00 hours sprinkle or 500 mg) once daily to 00:00 hours (e.g. aboutcapsules 23:00), or before sleep. If administered with food absorptionmay be delayed and the drug can be administered from approximately 20:30to 23:00 hours (e.g. about 22:00). Orfiril 150 to 750 mg (e.g. 300 22:00hours to 00:00 (e.g. enterotablets mg or 600 mg) once about 23.00) orbefore daily sleep. Orfiril Long 200 to 600 mg (e.g. 500 20:00 hours to00:00 (e.g. depot granules mg) once daily about 22:00) or before sleep.Ergenyl 200 to 600 mg (e.g. 300 21:00 hours to 00:00 (e.g. enterotabletsmg or 500 mg) once about 23:00) or before daily sleep. Ergenyl Retard100 to 750 mg (e.g. 250 22:00 to 01:00 (e.g. about depot granules mg or500 mg) once 23:00) or before sleep. daily Absenor 100-600 (e.g. 300 or500 21:00 to 00:00 (e.g. about enterotablets mg) once daily 23.00) orbefore sleep. If administered with food absorption may be delayed andthe drug can be administered from approximately 19:00 to 22:00 hours.Absenor tablets 300 to 600 mg (e.g. 300 23:00 to 01:00 (e.g. about mg)once daily 00.30) or before sleep. Convulex 150 to 600 mg (e.g. 30021:00 hours to 00:00 (e.g. capsules or 500 mg) once daily about 23:00)or before sleep. Epilim 100 to 600 mg (e.g. 400 21:00 to 00:00 (e.g.about gastroresistant or 500 mg) once daily 23.00) or before sleep.tablets Epilim Chrono/ 200 to 800 mg (e.g. 300 20:00 to 00:00 (e.g.about Depakine or 500 mg) once daily 22.00) or before sleep. ChronoEpilim 100 to 750 (e.g. 250 or 19:00 to 22:00 (e.g. about Chronospheres500 mg) once daily 20:30) or before sleep. Valprotek CR 300 to 600 mg(e.g. 300 19:00 to 22:00 (e.g. about or 500 mg) once daily 20.30) orbefore sleep. Depakene 250 to 750 mg (e.g. 250 21:00 hours to 00:00(e.g. capsules or 500 mg) once daily about 23:00) or before sleep.Depakene R 200 to 600 mg (e.g. 400 16:00 to 19:00 (e.g. about mg) oncedaily 17:30) Selenica R 200 to 600 mg (e.g. 400 9:00 to 12:00 (e.g.about mg) once daily 10:30) Episenta 150 to 750 mg (e.g 300 21:00 hoursto 00:00 (e.g. Prolonged or 600 mg) once daily. about 22:00) or beforerelease capsules sleep. Episenta 150 to 750 mg (e.g 300 21:00 hours to00:00 (e.g. Prolonged or 600 mg) once daily. about 22:00) or beforerelease granules sleep. Stavzor delayed 150 to 750 mg (e.g 300 23:00hours to 01:00 (e.g. release capsules or 600 mg) once daily. about23:30) or before sleep. If administered with food absorption may bedelayed and the drug can be administered from approximately 20:00 to23:00 hours. Valproic Acid 250 to 750 mg (e.g. 250 Approximately 22:00hours capsules, USP mg or 500 mg) once to 00:00 hours (e.g. about (Teva)daily 23:00), or before sleep. If administered with food absorption maybe delayed and the drug can be administered from approximately 19:00 to21:00 hours.

As used herein, references to the name of certain formulations willrefer to the corresponding formulation as sold/marketed in the relevantterritory (e.g. in the US, UK or Sweden) as on 1 Oct. 2014.

References in the above table to specific formulations by a specificname will include references to substantially identical formulationsthat may be referred to by another name (e.g. identical formulationssold and/or marketed using a different product name).

As described herein, the skilled person will understand thatadministration of a formulation to a patient with or shortly after foodmay delay release of the active ingredient and will be able to adjustthe time of administration accordingly. Unless otherwise stated,references herein to administration of a particular formulation at aparticular time (e.g. within a particular time period) will refer toadministration to the patient on an empty stomach.

Combination Treatments

Compounds of the invention may also be administered in combination with(e.g in a combined formulation with) other therapeutic agents that areuseful in the treating or preventing a pathological condition associatedwith excess fibrin deposition and/or thrombus formation.

In particular, pharmaceutical compositions as described in the eightaspect of the invention (including embodiments thereof) may comprisecompounds of the invention together with one or more pharmaceuticallyacceptable excipients and one or more other therapeutic agents that areuseful in the treating or preventing a pathological condition associatedwith excess fibrin deposition and/or thrombus formation.

In a particular embodiment of the first to seventh aspects of theinvention, the valproic acid or pharmaceutically acceptable salt thereofis administered in combination with one or more (e.g. one) othertherapeutic agents that are useful in the treating or preventing apathological condition associated with excess fibrin deposition and/orthrombus formation.

In a particular embodiment of the eight aspect of the invention, thepharmaceutical formulation further comprises one or more (e.g. one)other therapeutic agents that are useful in the treating or preventing apathological condition associated with excess fibrin deposition and/orthrombus formation. In such embodiments, the compounds of the inventionmay be provided in admixture with said one or more other therapeuticagent.

Thus, the skilled person will understand that the invention furtherprovides a process for the preparation of pharmaceutical formulations asdescribed herein (such as those described in the eight aspect of theinvention, including embodiments thereof), which process comprises thesteps of:

(a) bringing compounds of the invention into association with one ormore pharmaceutically acceptable excipient (e.g. to form an admixturethereof) and/or one or more (e.g. one) other therapeutic agents that areuseful in the treating or preventing a pathological condition associatedwith excess fibrin deposition and/or thrombus formation; and(b) formulating as a tablet or capsule (as described herein, e.g. withone or more coating).

As referred to herein, other therapeutic agents that are useful in thetreating or preventing a pathological condition associated with excessfibrin deposition and/or thrombus formation include: one or moreanti-thrombolytic agents; and/or one or more anticoagulant agents;and/or one or more antiplatelet agents; and/or one or more vasodilators,as known to those skilled in the art.

In particular embodiments, compounds of the invention may administeredand/or formulated in combination with:

-   -   one or more anti-platelet agents, including but not limited to        aspirin, persantin, ticagrelor and clopidogrel;    -   one or more anticoagulant agents, such as heparin, low molecular        weight heparin (LMWH), warfarin, anisindione, phenindone,        bishydroxycoumarin, bivalirudin, eptifibatid; one or more        vasodilators such as nitriles (for example, amylnitrile,        nitroglycerin, sodium nitrile, isosorbide dinitrate),        papaverine, nicotinic acid and cyclandelate.    -   one or more agents preventing cardiovascular events such as, but        not limited to statins, beta blockers, angiotensin converting        enzyme inhibitors, angiotensin II receptor antagonists or        diuretics; and/or    -   one or more anti-inflammatory agents including steroids and        NSAIDs (including but not limited to aspirin, ibuprofen,        naproxen and diclofenac);    -   one or more thrombolytic agents selected from, for example,        recombinant t-PA, prourokinase, urokinase or streptokinase.

In more particular embodiments, compounds of the invention may beadministered and/or formulated in combination with aspirin (i.e. atherapeutically effective amount of aspirin).

In yet more particular embodiments, compounds of the invention may beadministered and/or formulated in combination with clopidogrel (i.e. atherapeutically effective amount of Clopidogrel) or ticagrelor (i.e. atherapeutically effective amount of ticagrelor).

For the avoidance of doubt, the skilled person will understand that theterm “administered in combination with” includes concomitant, sequentialand separate administration. In this regard, sequential administrationmay refer to administration within the same therapeutic intervention(e.g. within one hour of the compound of the invention).

The skilled person will understand that references to an agent beingadministered in combination with another agent may also include akit-of-parts comprising the relevant agents (i.e. as separate componentswithin the same kit).

The skilled person will also understand that references to a first agentbeing administered in combination with a second agent will also thesecond agent being administered in combination the first agent, and soforth.

Patient Groups

The skilled person will understand that references herein to a “patient”will refer to living animals who may be subject to the treatment orprevention described herein. In particular, the term patient will referto a mammal. More particularly, the term patient will refer to a human(such an adult human).

Compounds of the invention may be particularly useful in the treatmentor prevention of (particularly, the prevention of) a pathologicalcondition associated with excess fibrin deposition and/or thrombusformation (such as those described herein) in patients at increased riskof developing one or more such condition.

In a particular embodiment of the first to seventh aspects of theinvention (including all embodiments thereof), the treatment orprevention (e.g. the prevention, which may also be referred to asprophylaxsis) is in a patient at increased risk of developing apathological condition associated with excess fibrin deposition and/orthrombus formation (which the skilled person will understand asreferring to reducing the risk of the relevant condition, as describedherein).

As described herein, several conditions and risk factors are associatedwith increased susceptibility to thrombotic events (i.e. thrombusformation). These include atherosclerosis, hypertension, abdominalobesity, smoking, sedentary lifestyle, and low-grade inflammation. Thus,in particular embodiments of the first to seventh aspects of theinvention (including all embodiments thereof), the treatment orprevention (e.g. the prevention, which may also be referred to asprophylaxsis) is in a patient having one or more such condition/riskfactor.

In more particular embodiments, the patient at increased risk ofdeveloping a pathological condition associated with excess fibrindeposition and/or thrombus formation is a patient who:

(i) is suffering from one or more medical condition associated withincreased risk of thrombus formation, such as metabolic syndrome (e.g.type II diabetes), oncologic diseases, heart failure, renal failureand/or sepsis;

(ii) has previously experienced one or more incidence of a pathologicalcondition associated with excess fibrin deposition and/or thrombusformation, such as one or more incidence of myocardial infarction,ischemic stroke and pulmonary embolism (e.g. one or more incidence ofischemic stroke, such as a major ischemic stroke, minor ischemic strokeor TIA); and/or(iii) has one or more lifestyle and/or environmental factors placingthem at said increased risk, such the patient being a smoker, obeseand/or having decreased mobility (e.g. the patient is bed-ridden, suchas a patient in a medical unit or elderly care unit).

Thus, in particular embodiments, references to a patient at increasedrisk of developing a pathological condition associated with excessfibrin deposition and/or thrombus formation will include references toan obese patient, e.g. a patient with a body mass index (BMI) that isabove 25 (e.g. above 30 and above 35).

As used herein, references to a patient at increased risk of developinga pathological condition associated with excess fibrin deposition and/orthrombus formation may also include patients (e.g. human male patients)who are 50 years of age or older (e.g. 60 years of age or older).

In particular embodiments, a patient at increased risk of developing apathological condition associated with excess fibrin deposition and/orthrombus formation may also be a patient who has elevated PAI-1 levels.

For example, as described herein, a patient at increased risk ofdeveloping a pathological condition associated with excess fibrindeposition and/or thrombus formation may also be a patient who issuffering from local or systemic inflammation, such as that associatedwith elevated PAI-1 levels.

Thus, in particular embodiments, a patient at increased risk ofdeveloping a pathological condition associated with excess fibrindeposition and/or thrombus formation may be a patient having PAI-1levels in morning plasma above about 20 ng/ml (e.g. above about 40ng/ml, such as above about 60 ng/ml, e.g. above about 80 ng/ml or, moreparticularly, above about 100 ng/ml).

For example, a patient at increased risk of developing a pathologicalcondition associated with excess fibrin deposition and/or thrombusformation may be a patient having PAI-1 levels in morning plasma aboveabout 20 ng/ml (e.g. above about 40 ng/ml, such as above about 60 ng/ml,e.g. above about 80 ng/ml or, more particularly, above about 100 ng/ml)and having experienced one or more incidence of myocardial infarction,ischemic stroke and pulmonary embolism (e.g. one or more incidence ofischemic stroke, such as a major ischemic stroke, minor ischemic strokeor TIA).

In certain embodiments, the patient is not suffering from a:

(i) a CNS or psychiatric disorder, such as epilepsy, migraine and/orbipolar disorder; and/or

(ii) Fragile X syndrome and/or familial adenomatous polyposis.

Thus, in a particular embodiment of the first to seventh aspects of theinvention (including all embodiments thereof), the treatment orprevention (e.g. the prevention) is in a patient who is:

(a) at increased risk of developing a pathological condition associatedwith excess fibrin deposition and/or thrombus formation (particularly asdefined herein); and

(b) is not suffering from a CNS or psychiatric disorder (as definedherein, particularly epilepsy and/or bipolar disorder).

FIGURES

FIG. 1 shows a schematic representation of the circadian rhythm (i.e.variation) of PAI-1 levels in an adult human during a typical 24 hourperiod. The lower curve represents the variation of PAI-1 levels in anormal (i.e. healthy) patient. The upper curve represents the variationin PAI-1 levels in a patient having increased levels of PAI-1 (e.g.patients with obesity and/or the metabolic syndrome). The y-axisrepresents arbitrary plasma levels and is abbreviated to illustrate thepositively skewed distribution toward high plasma levels inobesity/metabolic syndrome. The x-axis represents clock time.

EXAMPLES

The following examples are included to further illustrate the invention,although the skilled person will understand that the invention is in noway restricted to the specific aspects described therein.

Example 1—VPA and PAI-1

The effects of VPA on PAI-1 were analysed in two differentproof-of-concept studies in healthy subjects as well as in patients withmanifest atherosclerotic disease. The studies had a randomizedcross-over design and PAI-1 levels were investigated before and afterHDAC inhibition with valproic acid. PAI-1 plasma levels were measured inthe morning at the first day of the study as well as at the end of thetreatment period with VPA (see example 2 for details on the PAI-1analysis).

In the first study, 10 healthy non-smoking white male subjects (withmean BMI of approximately 26), aged 50-70 years were included andtreated with valproic acid 500 mg (Ergenyl Retard, Sanofi) twice dailyduring 14 days. Unexpectedly we detected a more than 50% reduction (from22.2 to 10.8 ng/ml, p<0.05) in circulating plasma PAI-1 levels duringmid-morning in comparison to the midmorning levels found beforetreatment with VPA.

In the second study, 16 non-smoking white male patients, aged 50-80years with a history of a myocardial infarction were included. On top oftheir ordinary prescription (beta-blocker, ACE-inhibitor, statin,aspirin) they were treated with valproic acid 500 mg (Ergenyl Retard,Sanofi), twice daily during 28 days. In this study we detected a 45%reduction in circulating plasma PAI-1 levels (from 19.6 ng/ml to 11ng/ml (p=0.01)), during midmorning.

Example 2—Intermediate Endpoint Study: Effects of Valproic Acid on InVivo PAI-1 in Man

An intermediate endpoint proof-of-concept study is performed in patientswith TIA/minor stroke investigated before and after treatment withValproic acid. Valproic acid is administrated as an enteric-coatedtablet with delayed absorption.

The study comprises 20 patients with TIA/minor stroke. Patients areinvestigated before and after oral treatment with 400 mg valproic acidonce time daily at 11 pm for 2 weeks. Plasma PAI-1 levels and plasmaconcentrations of valproic acid is followed daily during the studyperiod at the following time-points: 3 am, 6 am, 10 am, 16 pm, 22 pm PAMlevels are measured by commercially available ELISA-kits (Coaliza PAI-1,Chromogenix AB) and the plasma concentration of valproic acid anmetabolites thereof is analyzed according to clinical routine at theSahlgrenska University laboratory, Gothenburg, Sweden.

The plasma concentration of valproic acid is found to peak between 3 amand 6 am and thereafter declines to very low levels during the trough inPAI-1 concentrations. The peak in plasma valproic acid coincides withthe peak level of plasma PAI-1 between 3 am and 6 am. The plasmaconcentration of valproic acid and plasma PAI-1 levels follow each otherwith a pronounced circadian elevation with its peak during the earlymorning hours. The plasma PAI-1 levels are lowered by approximately 30%after the treatment.

Example 3—Clinical Outcome Study in High-Risk Patients for Prevention ofRecurrent Thromboembolic Events Using Valproic Acid

A clinical outcome study is performed in high-risk patients who haveexperienced a recent major atherothrombotic cardiovascular event(myocardial infarction or TIA/ischemic stroke) to investigate thepreventive effect of valproic acid treatment on the risk for recurrentevents. The annual risk for a recurrent atherothrombotic event in theinvestigated population is estimated to approximately 7%.

Patients are randomized in a parallel study design to receivedouble-blind oral treatment with 400 mg valproic acid (as in Example 2)or placebo once time daily at 11 pm, in addition to optimal conventionaltreatment. The event rate is monitored by Kaplan-Meyer statistics. Theprimary efficacy endpoint is the composite measure of either mortality,or non-fatal myocardial infarction or ischemic stroke. The study isevent-driven to a total of 180 events.

The study is expected to show that long-term valproic acid treatmentreduces this risk by approximately 30% in addition to that ofconventional therapy, i.e. lowers the annual absolute event rate toapproximately 5%. Thus, the study is expected to confirm the clinicalefficacy and feasibility of using valproic acid for secondary preventionof cardiovascular disease.

The invention claimed is:
 1. A method of treating or reducing the riskof a pathological condition associated with excess fibrin depositionand/or thrombus formation in a subject in need thereof, comprisingadministering to the subject at least one dose of a therapeuticallyeffective amount of valproic acid, or a pharmaceutically acceptable saltthereof, wherein the maximum plasma concentration (Cmax) of the valproicacid, or salt and/or metabolite thereof in the subject afteradministration occurs during a time period that is from four hoursbefore to one hour after the Cmax of PAI-1 in the subject.
 2. The methodof claim 1, wherein the at least one dose is administered during a timeperiod from about 8 pm to about 6 am.
 3. The method of claim 1, whereinthe time period is from about 2 am to about 7 am.
 4. The method of claim1, wherein the at least one dose of valproic acid, or thepharmaceutically acceptable salt thereof; is from about 50 to about 1000mg.
 5. The method of claim 1, wherein the at least one dose of valproicacid, or the pharmaceutically acceptable salt thereof; is from about 200mg to about 600 mg.
 6. The method of claim 1, wherein the at least onedose is a single dose.
 7. The method of claim 1, wherein the valproicacid, or a pharmaceutically acceptable salt thereof; is in the form of atablet or capsule for oral administration and at least 60% of thevalproic acid, or the pharmaceutically acceptable salt thereof, isreleased from the tablet or capsule during a time period from about fourto about eight hours after administration, or during a time period fromabout 2 am to about 6 am.
 8. The method of claim 1, wherein a seconddose is administered, and the second dose comprises about 10 mg to about500 mg of valproic acid, or a pharmaceutically acceptable salt thereof,which is administered about 10 hours to about 14 hours after the atleast one dose.
 9. The method of claim 1, wherein the valproic acid or apharmaceutically acceptable salt thereof is administered: (i) as asingle dose per 24 hour period (i.e., a single daily dose); and/or (ii)at a dose sufficient to achieve a reduction in PAI-1 plasma levels of atleast about 20%.
 10. The method of claim 1, wherein the pathologicalcondition associated with excess fibrin deposition and/or thrombusformation is selected from the group consisting of atherosclerosis,myocardial infarction, ischemic stroke, deep vein thrombosis,superficial vein thrombosis, thrombophlebitis, pulmonary embolism,disseminated intravascular coagulation, renal vascular disease andintermittent claudication.
 11. The method of claim 1, wherein thepathological condition associated with excess fibrin deposition and/orthrombus formation is ischemic stroke, and ischemic stroke comprisesmajor ischemic stroke and minor ischemic stroke.
 12. The method of claim1, wherein the pathological condition associated with excess fibrindeposition and/or thrombus formation is myocardial infarction.
 13. Themethod of claim 1, wherein the pathological condition associated withexcess fibrin deposition and/or thrombus formation is deep veinthrombosis.
 14. The method of claim 1, wherein the subject is a human.15. The method of claim 1, wherein the valproic acid, or apharmaceutically acceptable salt thereof, is in the form of a tablet orcapsule for oral administration and at least 70% of the valproic acid,or the pharmaceutically acceptable salt thereof, is released from thetablet or capsule during a time period from about four to about eighthours after administration, or during a time period from about 2 am toabout 6 am.
 16. A method of treating or reducing the risk of apathological condition associated with excess fibrin deposition and/orthrombus formation in a subject in need thereof, comprisingadministering to the subject a therapeutically effective dose ofvalproic acid, or a pharmaceutically acceptable salt thereof, during atime period from about 8 pm to about 6 am, wherein the maximum plasmaconcentration (Cmax) of the valproic acid, or salt and/or metabolitethereof in the subject after administration occurs during a time periodthat is from four hours before to one hour after the Cmax of PAI-1 inthe subject.
 17. The method of claim 16, wherein the valproic acid, or apharmaceutically acceptable salt thereof, is in the form of a tablet orcapsule for oral administration, and substantially all of the valproicacid, or a pharmaceutically acceptable salt thereof, is released fromthe tablet or capsule during a time period from about 2 am to about 6am.
 18. The method of claim 16, wherein a single dose of valproic acid,or a pharmaceutically acceptable salt thereof, is administered to asubject in a 24 hour period, wherein the dose of valproic acid, or apharmaceutically acceptable salt thereof, is from about 50 mg to about1000 mg, or from about 200 mg to about 600 mg.
 19. The method of claim16, wherein the valproic acid or a pharmaceutically acceptable saltthereof is administered: (i) as a single dose per 24 hour period (i.e.,a single daily dose); and/or (ii) at a dose sufficient to achieve areduction in PAI-1 plasma levels of at least about 20%.
 20. The methodof claim 16, wherein the pathological condition associated with excessfibrin deposition and/or thrombus formation is selected from the groupconsisting of atherosclerosis, myocardial infarction, ischemic stroke,deep vein thrombosis, superficial vein thrombosis, thrombophlebitis,pulmonary embolism, disseminated intravascular coagulation, renalvascular disease and intermittent claudication.
 21. The method of claim16, wherein the pathological condition associated with excess fibrindeposition and/or thrombus formation is ischemic stroke, and ischemicstroke comprises major ischemic stroke and minor ischemic stroke. 22.The method of claim 16, wherein the subject is a human.
 23. The methodof claim 16, wherein the pathological condition associated with excessfibrin deposition and/or thrombus formation is myocardial infarction.24. The method of claim 16, wherein the pathological conditionassociated with excess fibrin deposition and/or thrombus formation isdeep vein thrombosis.